JP2012254055A - Method for collecting nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for directly collecting a nucleic acid from biological samples such as feces or the like, reducing carryover of impurities originated from the biological samples, and collecting nucleic acid with high purity.SOLUTION: The method for collecting nucleic acid from biological samples is characterized by comprising: (A) a process of mixing the biological samples with a cytolytic agent to prepare a suspension; (B) a process of centrifugally treating the suspension prepared in the process (A) to collect supernatant as a crude nucleic acid solution; and (C) a process of collecting nucleic acid from the crude nucleic acid solution collected in the process (B), wherein a treatment of bringing into contact with chemical fibers or natural fibers (excluding fibers of silica compounds) is applied to the suspension prepared in the process (A) before the process (B), to the crude nucleic acid solution collected in the process (B) before the process (C), and to the collected nucleic acid after the process (C).

Description

  The present invention relates to a method for recovering highly pure nucleic acid from a biological sample.

  In recent years, as a new non-invasive, simple and reliable test method suitable for regular medical checkups, the presence or absence of cancer cells or the presence of cancer cell-derived genes in biological samples such as feces Examination to examine is attracting attention. These examination methods are expected to be highly reliable examination methods because they directly examine the presence or absence of cancer cells or cancer cell-derived genes.

  On the other hand, a biological sample usually contains various substances, and substances having an inhibitory action on nucleic acid amplification reactions such as PCR (Polymerase Chain Reaction) are also included. For example, it is known that bile acids and salts thereof contained in a large amount in feces have an inhibitory effect on nucleic acid amplification reactions such as PCR (see, for example, Non-Patent Document 1). The average amount of excreted feces for adults is about 200 to 400 g / day, but there is a report that 200 to 650 mg / day of bile acids are excreted in the feces of healthy people. That is, when converted per gram of stool, about 0.5 mg to 3.25 mg for healthy persons and 10 times as much bile acids for patients. There are reports that the bile salt inhibits the PCR effect at a concentration of about 50 μg / mL. When nucleic acid is extracted from stool and amplified by PCR or the like, inhibition of nucleic acid amplification reaction such as bile salt is inhibited. It is desirable to prevent material carryover.

Wilson IG, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63, 3741-3751.

  In feces, there are not only nucleic acid amplification reaction inhibitors such as bile acids but also many impurities such as contaminants, undigested materials and fibers, which adversely affect the recovery, amplification and detection of nucleic acids. Probability is high. Therefore, when recovering RNA directly from stool without separating the cell fraction, not only nucleic acid amplification reaction inhibitors, but also proteins and metabolites that elute during cell lysis are removed, and these impurities are carried. It is desirable to prevent overshoot.

  However, Non-Patent Document 1 discloses impurities in nucleic acid amplification reaction inhibitors such as bile acids and bile salts in feces, proteins and metabolites eluted during cell lysis, and impurities such as debris when nucleic acids are collected from feces. There is no mention of preventing carryover.

  An object of the present invention is to provide a method for directly recovering nucleic acid from a biological sample such as stool, which can reduce the carryover of impurities derived from the biological sample and recover high-purity nucleic acid.

  As a result of intensive studies to solve the above problems, the present inventors have suspended biological samples such as feces with a cell lysing agent, and then removed insoluble components from the obtained suspension, and the remaining crude nucleic acid. In a method for recovering nucleic acid from a solution, insoluble components are removed from the suspension by centrifugation, and at any point from the preparation of the suspension to the recovery of the nucleic acid, a liquid fraction containing nucleic acid (for example, , Suspension, crude nucleic acid solution, or recovered nucleic acid) is brought into contact with chemical fiber or natural fiber (excluding silica compound fiber), thereby carrying out impurity carryover to the recovered nucleic acid. The present invention has been completed by finding that it can be reduced.

That is, the present invention
(1) A method for recovering nucleic acid from a biological sample,
(A) mixing a biological sample and a cell lysing agent to prepare a suspension;
(B) centrifuging the suspension prepared in the step (A) and recovering the supernatant as a crude nucleic acid solution;
(C) recovering nucleic acid from the crude nucleic acid solution recovered in the step (B),
Have
A treatment in which chemical fiber or natural fiber (excluding silica compound fiber) is contacted,
Before the step (B), with respect to the suspension prepared by the step (A),
The nucleic acid recovery, which is performed on the crude nucleic acid solution recovered in the step (B) before the step (C) or on the recovered nucleic acid after the step (C). Method,
(2) The method for recovering nucleic acid according to (1) above, wherein the chemical fiber or natural fiber forms a porous body,
(3) The method for recovering nucleic acid according to (2) above, wherein the chemical fiber or natural fiber has an average pore diameter of 1 to 600 μm,
(4) The method for recovering nucleic acid according to any one of (1) to (3), wherein the chemical fiber or natural fiber is a nonwoven fabric,
(5) The method for recovering nucleic acid according to any one of (1) to (4), wherein the suspension contains an RNase inhibitor,
(6) The method for recovering a nucleic acid according to any one of (1) to (5), wherein the centrifugal force of the centrifugation treatment is 2000 × g or more,
(7) The method for recovering nucleic acid according to any one of (1) to (6), wherein the biological sample is feces.
(8) A method for analyzing a nucleic acid in a biological sample,
(A) mixing a biological sample and a cell lysing agent to prepare a suspension;
(B) centrifuging the suspension prepared in the step (A) and recovering the supernatant as a crude nucleic acid solution;
(C) recovering nucleic acid from the crude nucleic acid solution recovered in the step (B),
(D) analyzing the nucleic acid recovered in the step (C);
Have
A treatment in which chemical fiber or natural fiber (excluding silica compound fiber) is contacted,
Before the step (B), with respect to the suspension prepared by the step (A),
Nucleic acid analysis, which is performed on the crude nucleic acid solution recovered in the step (B) before the step (C) or on the recovered nucleic acid after the step (C). Method,
(9) The nucleic acid analysis method according to (8), wherein in the step (D), a nucleic acid derived from a mammalian cell is analyzed,
(10) The nucleic acid-derived nucleic acid analysis method according to (9), wherein the mammalian cell-derived nucleic acid is a marker indicating neoplastic conversion or a marker indicating inflammatory digestive tract disease,
(11) In the step (D), a reverse transcription reaction is performed using the RNA recovered in the step (C) as a template, and the obtained cDNA is used to indicate a marker for neoplastic transformation or inflammatory digestive organ disease A method of analyzing a nucleic acid according to (8) or (9), wherein a marker indicating
(12) A kit used for recovering nucleic acid from a biological sample,
A kit for nucleic acid recovery, comprising a cell lysing agent and chemical fibers or natural fibers (excluding silica compound fibers);
(13) The nucleic acid recovery kit according to (12), wherein the chemical fiber or natural fiber is a nonwoven fabric,
Is to provide.

  According to the method for recovering nucleic acid of the present invention, it is possible to recover nucleic acid with high purity with less impurity carryover. For this reason, the method for recovering nucleic acid of the present invention is particularly suitable for recovering nucleic acid from a biological sample containing a relatively large amount of contaminants such as feces.

In Example 1, it is the figure which showed the result of having calculated the relative value of the expression level of xenoRNA gene. In Example 2, it is the figure which showed the result of having calculated the relative value of the expression level of xenoRNA gene. In Example 3, it is the figure which showed the result of having measured the amount of RNA collect | recovered from each stool specimen AC. In Example 3, it is the figure which showed the result of having calculated the expression level (measured value of fluorescence intensity) of (beta) 2M gene. In Example 4, it is the figure which showed the result of having calculated the relative value of the expression level of xenoRNA gene.

  In the present specification, an inhibitory substance means a substance that acts inhibitory to an enzymatic reaction using a nucleic acid as a substrate. The enzyme reaction is not particularly limited as long as it is an enzyme reaction using a nucleic acid as a substrate, and examples thereof include enzyme reactions generally used in nucleic acid analysis such as reverse transcription reaction and nucleic acid synthesis reaction. Specific examples of the inhibitor include bile acids and bile salts. In addition, proteins, metabolites, debris, and the like that are eluted when cells in a biological sample are lysed are also included as inhibitors.

  Here, the nucleic acid synthesis reaction means a reaction involving nucleic acid elongation by polymerase or ligase. Examples of the reaction accompanied by nucleic acid extension by polymerase include PCR (polymerase chain reaction), real-time PCR, SDA (Standard Displacement Amplification) and the like. Examples of the reaction accompanied by nucleic acid elongation by ligase include LCR (ligase chain reaction).

  In the present specification, the cell includes not only mammalian cells such as human-derived cells but also bacteria such as bacteria.

<Nucleic acid recovery method>
The nucleic acid recovery method of the present invention (hereinafter sometimes referred to as “recovery method of the present invention”) is a method of recovering nucleic acid from a biological sample, comprising the following steps (A) to (C): And the process which makes a chemical fiber or a natural fiber (however, except a silica compound fiber) contact is the following process (C) with respect to the suspension prepared by the following process (A) before the following process (B). ) Is performed on the crude nucleic acid solution collected in the following step (B), or on the collected nucleic acid after the following step (C).
(A) mixing a biological sample and a cell lysing agent to prepare a suspension;
(B) centrifuging the suspension prepared in the step (A) and recovering the supernatant as a crude nucleic acid solution;
(C) A step of recovering nucleic acid from the crude nucleic acid solution recovered in the step (B).

  Examples of biological samples provided for the recovery method of the present invention include feces, urine, blood, bone marrow fluid, lymph fluid, sputum, saliva, semen, bile, pancreatic fluid, ascites, exudate, amniotic fluid, intestinal lavage fluid, and lung lavage fluid. , Bronchial lavage fluid, or bladder lavage fluid. In addition, cultures such as cultured cells may be used. The biological sample used in the recovery method of the present invention is particularly preferably a stool, blood, or culture such as cultured cells. The biological sample is not particularly limited as long as it is collected from a living organism, but is preferably derived from a mammal, and more preferably derived from a human. For example, it is preferably a biological sample collected from a human for periodic medical examination or diagnosis, but it may be a biological sample such as livestock or wild animals. Moreover, although it may be preserved for a certain period after collection, it is preferably immediately after collection. When the biological sample is stool, the stool used in the recovery method of the present invention is preferably immediately after excretion, but it may be one that has passed time after excretion.

  The amount of the biological sample used in the recovery method of the present invention is not particularly limited, and can be determined as appropriate in consideration of the type of biological sample, the method for analyzing the nucleic acid recovered from the biological sample, and the like. . For example, in the case of feces, it is preferably 10 mg to 1 g. If the amount of stool becomes too large, it will take time and effort to collect the stool and the size of the stool collection container will increase. On the other hand, if the amount of stool is too small, the number of mammalian cells such as colon exfoliated cells contained in the stool becomes too small, so that the required amount of nucleic acid cannot be recovered and the accuracy of the target nucleic acid analysis is reduced. There is a fear. In addition, stool is heterogeneous, that is, since various components are present unevenly, it is preferable to collect from a wide range of stool at the time of stool collection in order to avoid the influence of the localization of mammalian cells. .

Hereinafter, each process will be described.
First, as step (A), a biological sample and a cell lysing agent are mixed to prepare a suspension. By suspending the biological sample in a cell lysing agent, nucleic acid is extracted from solid components such as cells in the biological sample.

  The cell lysing agent is not particularly limited as long as it has an action of eluting nucleic acid contained in the cell to the outside of the cell, and it is appropriately selected from compounds and compositions known in the technical field. It can be selected and used. Specific examples of the compound or the like used as a cell lysing agent include compounds usually used as a protein denaturant such as an organic solvent, a chaotropic salt, and a surfactant. In addition, as a cytolytic agent, one type of compound may be added and two or more types of compounds may be added.

The organic solvent used as the cell lysing agent is preferably phenol. Phenol may be neutral or acidic. When acidic phenol is used, RNA can be selectively extracted into the aqueous layer rather than DNA.
Moreover, the phenol used as a cell lysis agent may be added to a biological sample by phenol alone, or may be added to a biological sample as a phenol mixture including other components that do not inhibit the cell lysis action by phenol. However, it is preferable that the phenol mixture does not contain chloroform.

  Examples of the chaotropic salt used as the cell lysing agent include guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, sodium perchlorate, and sodium trichloroacetate.

  The surfactant used as the cell lysis agent may be an ionic surfactant, but is preferably a nonionic surfactant. Examples of the nonionic surfactant include Tween 80, CHAPS (3- [3-colamidopropyldimethylammonio] -1-propanesulfonate), Triton X-100, Tween 20, and the like.

  If the biological sample has a large amount of liquid such as urine or the cell lysing agent is a liquid such as phenol, a suspension should be prepared by adding the cell lysing agent directly to the biological sample. Can do. On the other hand, when mixing a biological sample with a high solid content and a solid cell lysing agent, a cell lysing agent solution is prepared by dissolving the cell lysing agent in advance in an appropriate buffer, and the cell lysing agent solution and the biological sample are mixed. By mixing, a suspension can be prepared. As the buffer for dissolving the cell lysing agent, for example, a phosphate buffer such as PBS, a Tris buffer, a citrate buffer, or the like can be used.

Furthermore, the suspension prepared in the step (A) may contain a nucleic acid degradation inhibitor such as an RNase inhibitor in order to improve the storage stability of the nucleic acid derived from the biological sample. Examples of RNase inhibitors include chelating agents such as EDTA, chaotropic salts, and the like.
In addition, you may add a coloring agent suitably to the suspension liquid prepared in a process (A). By adding a colorant to the suspension, effects such as prevention of accidental ingestion and relaxation of the color of the biological sample can be obtained. The colorant is preferably a colorant used as a food additive, and is preferably blue or green. Examples include Fast Green FCF (Green No. 3), Brilliant Blue FCF (Blue No. 1), Indigo Carmine (Blue No. 2), and the like. In addition, a plurality of colorants may be mixed and added, or may be added alone.

  In the case of a biological sample having a large amount of solid components such as feces, the volume of a cell lysing agent solution or a cell lysing agent (hereinafter simply referred to as a cell lysing agent solution) mixed with the collected biological sample is particularly limited. Although it is not a thing, it is preferable that the mixing ratio of a biological sample and a cell lysis agent solution has a cell lysis agent solution volume of 1 or more with respect to a biological sample volume of 1. By using an amount of cell lysing agent solution equal to or more than that of the biological sample, the biological sample and the cell lysing agent can be quickly mixed, and the cell lysing agent can sufficiently act on the biological sample. is there. In particular, the biological sample can be quickly and effectively dispersed in the cell lysing agent solution by mixing the cell lysing agent solution with a volume of 5 times or more with respect to the biological sample. It is also possible to suppress the influence of the decrease in the concentration of the cytolytic agent due to the water contained in the. On the other hand, it is often preferable that the total amount of the mixture of the biological sample and the cell lysing agent solution is relatively small because handling becomes easy. For example, when stool is used, stool can be collected in a stool collection container provided with a cell lysing agent solution in advance, and a mixture can be prepared in the container. When the amount of the solution is equal, the stool collection container containing the cell lysing agent solution can be reduced in weight and size. Thus, since it becomes possible to improve the handleability of the biological sample and the resulting suspension and the dispersibility of the biological sample in the cell lysing agent solution in a well-balanced manner, the biological sample is feces. More preferably, the mixing ratio of the biological sample and the cell lysing agent solution is 1: 1 to 1:20, more preferably 1: 3 to 1:10, and more preferably about 1: 5. preferable.

  The concentration of the cell lysing agent in the suspension obtained by mixing the biological sample and the cell lysing agent solution is the concentration at which the nucleic acid can be eluted from the solid component in the biological sample (the concentration that exerts the nucleic acid elution effect). ) As long as it is not particularly limited and can be appropriately determined in consideration of the amount of biological sample, the subsequent nucleic acid recovery / analysis method, and the like.

  A method for preparing a suspension by mixing a biological sample and a cell lysing agent solution is not particularly limited as long as it is a method of mixing by a physical method. For example, the collected biological sample may be put into a sealable container in which a cell lysing agent solution has been put in advance and sealed, and then mixed by turning the container upside down. You may mix by applying to shakers, such as a vortex. Further, the biological sample and the cell lysing agent solution may be mixed in the presence of the mixing particles. Since they can be mixed quickly, a method using a shaker or a method using mixing particles is preferable. In particular, by using a collection container that contains particles for mixing in advance, it can be quickly mixed even in an environment without a special device such as a home.

  The particle for mixing is a composition that does not impair the nucleic acid elution effect of the cell lysing agent solution, such as hardness that can promote lysis of cells in the biological sample by the cell lysing agent by hitting the biological sample such as feces. The particles are not particularly limited as long as they have specific gravity and have sufficiently low affinity for the eluted nucleic acid, and may be particles made of one kind of material, and may be made of two or more kinds of materials. The particle | grains which become may be sufficient. Examples of such mixing particles include particles made of plastic, latex, metal, and the like. In addition, the mixing particles may be magnetic particles or non-magnetic particles.

  Next, as the step (B), the suspension prepared in the step (A) is centrifuged, and the supernatant is recovered as a crude nucleic acid solution. Since many biological samples have a high water content, when an organic solvent such as phenol is used as a cell lysing agent, the suspension is separated into a lower organic solvent layer and an upper aqueous layer by centrifugation. Divided. In this case, the upper aqueous layer is recovered as a supernatant.

  The conditions for the centrifugation treatment are not particularly limited as long as the insoluble solid component in the suspension can be separated from the aqueous liquid component, and the kind and amount of the biological sample and the cell lysing agent are not limited. It can be adjusted as appropriate in consideration of the above. For example, the centrifugation treatment may be performed at room temperature or in a low temperature environment such as 4 ° C. For example, when phenol is used as the cell lysing agent, the phenol layer, the insoluble solid content layer, and the aqueous layer can be sufficiently separated if the centrifugal force is 2000 × g or more. The upper limit value of the centrifugal force is not particularly limited, but is preferably not excessively large. For example, if it is 12000 * g or less, since a large centrifuging apparatus is not required, an apparatus capable of automatically executing the entire process of the recovery method of the present invention can be easily realized.

  In the present invention, by treating a biological sample with a cell lysing agent, nucleic acid is extracted from a cell or the like contained in the biological sample into a solution, and then a biological component insoluble in water is removed by centrifugation. Remove. By using the obtained supernatant as a crude nucleic acid solution and recovering the nucleic acid from this crude nucleic acid solution, carryover of an inhibitory substance derived from a biological sample can be significantly reduced. For this reason, by using the nucleic acid collected from the biological sample by the collection method of the present invention for the analysis, the influence of the inhibitor is reduced, and a highly reliable analysis result can be obtained.

  Conventionally, phenol is added to and mixed with a biological sample, and chloroform is further added and mixed, followed by centrifugation to recover the supernatant, and nucleic acid is recovered from the obtained supernatant. It has been broken. On the other hand, when phenol is used as a cell lysing agent in the recovery method of the present invention and chloroform treatment is performed as a crude purification treatment before the subsequent step (C) in step (B), phenol is added. The mixture thus obtained is centrifuged, and chloroform is added to the obtained supernatant to obtain a supernatant, and nucleic acid is recovered from the supernatant. Thus, it is not clear why the carry-over of the inhibitory substance can be significantly reduced by performing the centrifugation process in advance before adding chloroform, but by performing the centrifugation process in two steps, It is presumed that not only the inhibitory substance but also impurities such as proteins and metabolites eluted during cell lysis can be efficiently precipitated and removed. In addition, by centrifugation after phenol treatment, it is possible to effectively extract and remove insoluble substances as well as high-solubility inhibitory substances in phenol, followed by further chloroform treatment, thereby inhibiting high solubility in chloroform. It may be inferred that the substance can be effectively extracted and removed.

  Thereafter, as step (C), the nucleic acid is recovered from the crude nucleic acid solution recovered in step (B). The nucleic acid recovery method is not particularly limited, and can be performed by a known method such as an ethanol precipitation method or a cesium chloride ultracentrifugation method. For example, the nucleic acid can be recovered by adding an inorganic support to the crude nucleic acid solution, adsorbing the eluted nucleic acid to the inorganic support, and then eluting the nucleic acid from the inorganic support. As the inorganic support for adsorbing nucleic acid, a known inorganic support capable of adsorbing nucleic acid can be used. The shape of the inorganic support is not particularly limited, and may be in the form of particles or a film. Examples of the inorganic support include silica-containing particles (beads) such as silica gel, siliceous oxide, glass, and diatomaceous earth. Solvents for eluting adsorbed nucleic acids from inorganic supports are usually used to elute nucleic acids from these known inorganic supports in consideration of the type of nucleic acid to be recovered, the subsequent nucleic acid analysis method, and the like. A solvent can be appropriately used. The elution solvent is particularly preferably purified water. In addition, before eluting nucleic acid, it is preferable to wash | clean the inorganic support body which adsorbed the nucleic acid using a suitable washing buffer.

  In the present invention, at any point after step (B), the liquid fraction containing the nucleic acid is brought into contact with chemical fibers or natural fibers (excluding silica compound fibers). In addition to centrifugation, impurities such as inhibitors are brought into contact with the fibers and adsorbed to remove impurities from the liquid fraction. As a result, impurities carry over the nucleic acid recovered from the sample. Can be further reduced, and nucleic acids with higher purity can be recovered.

  The liquid fraction containing the nucleic acid specifically includes the suspension prepared in step (A), the crude nucleic acid solution recovered in step (B), and the nucleic acid recovered in step (C). Examples include solutions. That is, in the present invention, chemical fiber or the like is brought into contact with the suspension prepared in step (A) before step (B) or recovered in step (B) before step (C). A chemical fiber or the like is brought into contact with the prepared crude nucleic acid solution, or after the step (C), a chemical fiber or the like is brought into contact with the recovered nucleic acid, and then the fiber and the nucleic acid are separated.

  The fibers used in the recovery method of the present invention are fibers other than silica compound fibers, and examples thereof include polypropylene, polyethylene, nylon, rayon, acrylic, and vinylon. Examples of the natural fiber used in the recovery method of the present invention include cellulose, cotton fiber, pulp, and carbon fiber. In addition, the fiber brought into contact with the liquid fraction containing the nucleic acid may be only one type of fiber, or a combination of two or more types of fibers.

  The form of the fiber to be brought into contact with the liquid fraction containing the nucleic acid is not particularly limited as long as it is a porous body having an average pore diameter within a predetermined range, and is in the form of a sheet such as a cloth, a membrane, or a filter. It may be a hollow fiber or a bead (particle). The fabric may be a woven fabric or a non-woven fabric. In the present invention, a nonwoven fabric is preferable because a wide variety of impurities can be adsorbed efficiently.

  The pore size of the fiber is large enough to adsorb inhibitors such as bile acids and salts thereof, proteins and metabolites eluted when cells are dissolved, debris, and the like. When the average pore diameter of the fiber is too small, the liquid fraction containing the nucleic acid hardly penetrates into the fiber, so that there is a possibility that the adsorption removal of the inhibitor is insufficient. On the other hand, when the average pore diameter of the fibers is too large, the amount of fibers per unit area with which the liquid fraction containing the nucleic acid comes into contact is small, which may reduce the adsorption removal efficiency of the inhibitor. When the average pore diameter of the fibers is 1 to 600 μm, the permeability of the liquid fraction containing nucleic acid is high, and adsorption and removal of inhibitors and the like can be performed efficiently. The average pore diameter of the fibers used in the present invention is preferably 5 to 600 μm, more preferably 10 to 600 μm, and further preferably 100 to 600 μm. In the present invention, among them, a fiber having a pore size of 1 to 600 μm is preferable, and a nonwoven fabric having a pore size of 1 to 600 μm is particularly preferable.

  In the present invention and the present specification, a nonwoven fabric is a sheet-like or web structure made by bonding or entanglement of short fibers or filaments using mechanical, thermal, or chemical means ("No. 2nd Edition Textile Handbook ", edited by Textile Society, published by Maruzen Co., Ltd. Nonwoven fabrics are produced by various methods, but the basic processes are a web forming process (a sheet of fiber lump in which fibers are aligned to some extent), a web bonding process, and a finishing process. Various types of fibers are used for nonwoven fabrics, from natural fibers to chemical fibers. Commonly used are cotton, rayon, polyester, polypropylene, nylon, and other silica compounds such as acrylic, vinylon, and glass. Fiber, pulp, carbon fiber, etc. are also used. The method of forming the web is roughly classified into wet type, dry type and direct type. The direct theory method is also called a direct spinning method, and is a process in which fibers spun from a molten polymer solution are collected into a direct web. Examples of the method include a spunlace method, a spunbond method, a melt blow method, a needle punch method, and a stitch bond method. In the present invention, a nonwoven fabric obtained from any method may be used.

In the present invention, the method of bringing the liquid fraction containing nucleic acid into contact with the fiber is not particularly limited. For example, the fiber may be suspended on the liquid surface of the liquid fraction containing nucleic acid, The fibers may be immersed in the solution containing the liquid fraction, the liquid fraction containing the nucleic acid may be allowed to flow through the surface of the sheet-like fiber such as a nonwoven fabric, and the liquid fraction containing the nucleic acid may be allowed to flow through. May pass through a non-woven fabric filter or may be filtered with a filter paper. In addition, the liquid fraction containing nucleic acid may be passed through a chip filled with fibers inside, or the liquid fraction containing nucleic acid may be passed through a chip whose surface is coated with fibers. When the liquid fraction containing nucleic acid is permeated through a nonwoven fabric filter, filter paper, chip, etc., the obtained permeate or filtrate is a liquid fraction containing nucleic acid after being brought into contact with a chemical fiber or the like. .
Moreover, the chemical fiber etc. which were made to contact the liquid fraction containing a nucleic acid are removed after that. For example, after putting a fiber piece such as a non-woven fabric fragment into a liquid fraction containing nucleic acid, performing a centrifugal separation process, removing the fiber piece by precipitation, and contacting the obtained supernatant with a chemical fiber or the like The liquid fraction containing the nucleic acid of In addition, after the nonwoven fabric is suspended on the liquid surface containing the nucleic acid, the nonwoven fabric may be separated from the solution and recovered.

  The liquid fraction containing nucleic acid is the crude nucleic acid solution recovered in the step (B), which is subjected to chloroform treatment as a crude purification treatment before the subsequent step (C) in the step (B), and the chemical fiber Or the like is a fiber piece or the like, the raw nucleic acid solution first charged with the fiber piece or the like can be subjected to a chloroform treatment. The fiber pieces are separated and removed together with other insoluble substances by the centrifugal separation after mixing with chloroform.

  The nucleic acid recovered by the recovery method of the present invention can be analyzed using a known nucleic acid analysis method in the same manner as nucleic acids recovered by other methods. In particular, it is preferably used for nucleic acid analysis for examining the presence or absence of onset of cancer or infectious disease, which is strongly demanded for early detection. In addition, by examining whether the nucleic acid derived from the pathogen causing the infection or the like, for example, a nucleic acid derived from a virus, a nucleic acid derived from a parasite, a nucleic acid derived from a bacterium, or the like is detected from the collected nucleic acid, It is possible to examine the presence or absence of infectious diseases and the presence or absence of parasites of living organisms from which biological samples have been collected.

  When stool is used as a biological sample, the collected nucleic acid is preferably used for analysis of nucleic acid derived from gastrointestinal cells such as the large intestine, small intestine, stomach, etc., and used for analysis of nucleic acid derived from colon exfoliated cells. It is particularly preferred that For example, the nucleic acid recovered by the recovery method of the present invention is a nucleic acid analysis for examining the presence or absence of onset of colorectal cancer or colorectal adenoma and the presence or absence of inflammatory diseases such as colitis, enterocolitis, gastritis, pancreatitis, etc. Can be used. In addition, it may be used for examination of raised lesions such as polyps and examination of diseases of the large intestine such as gastric ulcer, small intestine, stomach, liver, gallbladder, and bile ducts.

<Nucleic acid recovery kit>
By collecting the cell lysing agent and the fiber into a kit, the recovery method of the present invention can be performed more simply. In the present invention, a kit including a non-woven fabric as a fiber is preferable, and a non-woven fabric made of polypropylene or polyethylene is more preferable. In addition to the cell lysing agent and the fiber, the nucleic acid recovery kit may include other reagents used in the recovery method of the present invention. For example, a buffer for diluting / dissolving a cell lysing agent, chloroform used for protein removal, a reagent for recovering nucleic acid from a suspension or a crude nucleic acid solution, and the like can be mentioned. Moreover, it can also be set as the nucleic acid collection kit of this invention by adding a fiber to commercially available kits, such as a nucleic acid extraction kit using a cell lysing agent.

<Nucleic acid analysis method>
The nucleic acid analysis method of the present invention (hereinafter sometimes referred to as “the analysis method of the present invention”) is characterized by analyzing the nucleic acid recovered by the recovery method of the present invention. Specifically, after the steps (A) to (C), a step of analyzing the nucleic acid recovered in the step (C) is performed as a step (D).

  The method for analyzing the nucleic acid in step (D) is not particularly limited, and may be appropriately selected from known analysis methods in consideration of the type of nucleic acid to be analyzed, the purpose of analysis, and the like. it can. Examples of the analysis method include a method for quantifying a nucleic acid and a method for detecting a specific base sequence region using PCR or the like. In addition, when RNA is recovered, cDNA can be synthesized by reverse transcription and used for analysis in the same manner as DNA using the cDNA. When DNA is used among nucleic acids collected from a biological sample, for example, mutation analysis or epigenetic change analysis on DNA can be performed. Examples of mutation analysis include analysis of base insertion, deletion, substitution, duplication, or inversion. Examples of epigenetic change analysis include analysis of methylation and demethylation. Moreover, the presence or absence of the onset of cancer can be investigated by detecting the presence or absence of a genetic variation such as a base sequence region containing microsatellite. On the other hand, when RNA is used among nucleic acids recovered from biological samples, for example, mutations such as base insertion, deletion, substitution, duplication, inversion, or splicing variant (isoform) on RNA Can be detected. In addition, functional RNA (non-coding RNA) analysis, for example, analysis of transfer RNA (transfer RNA, tRNA), ribosomal RNA (ribosomal RNA, rRNA), microRNA (miRNA, microRNA) and the like can be performed. In addition, the RNA expression level can be detected and analyzed. In particular, it is preferable to perform mRNA expression analysis, K-ras gene mutation analysis, DNA methylation analysis, and the like. These analyzes can be performed by methods known in the art. Commercially available analysis kits such as a K-ras gene mutation analysis kit and a methylation detection kit may also be used.

  In the analysis method of the present invention, nucleic acid derived from any species contained in a biological sample may be detected depending on the purpose, but it is preferable to detect and analyze nucleic acid derived from a mammalian cell. Further, when stool is used as a biological sample, it is preferable to detect and analyze nucleic acid derived from digestive tract cells or colon exfoliated cells of mammals excreting the stool.

  In particular, it is preferably subjected to analysis for detecting a marker indicating neoplastic transformation or a marker indicating inflammatory digestive organ disease. Examples of markers indicating neoplastic conversion include known cancer markers such as carcinoembryonic antigen (CEA) and sialyl Tn antigen (STN), and mutations such as APC gene, p53 gene, and K-ras gene. There is presence or absence. In addition, detection of methylation of genes such as p16, hMLHI, MGMT, p14, APC, E-cadherin, ESR1, and SFRP2 is also useful as a diagnostic marker for colorectal diseases (for example, Lind et al., “A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines ", Molecular Cancer, 2004, Vol. 3, Chapter 28). In addition, it has already been reported that DNA derived from Helicobacter pylori in stool samples can be used as a marker for gastric cancer (for example, Nilsson et al., Journal of Clinical Microbiology, 2004, Vol. 42, No. 8, No. 1). (See pages 3781-8.) On the other hand, examples of markers that indicate inflammatory digestive tract diseases include Cox-2 gene-derived nucleic acids.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
After collecting stool from two healthy individuals (A, B), about 0.5 g each was taken into 12 15 mL tubes as quickly as possible, and snap-frozen using liquid nitrogen at −80 ° C. saved. Among the stool collected from each healthy person, an RNA solution was prepared from the stool contained in each of three 15 mL tubes by the following collection methods 1 to 4.
Note that a phenol mixture was used as a cell lysing agent, and a Sanispec test bag with a filter (manufactured by ASONE) was used as a nonwoven fabric filter. The filtrate which permeate | transmitted this Sanispec test bag with a filter is normally used for a bacteria and cell test | inspection.

Recovery method 1 (in the case of no centrifugation treatment and no nonwoven fabric filter treatment):
To the frozen stool, a phenol mixture “Trizol” (manufactured by Invitrogen) was added and dissolved sufficiently, and then chloroform was added and mixed well using a vortex. Thereafter, centrifugation was performed at 12,000 × g and 4 ° C. for 20 minutes. RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. Specifically, sodium acetate and 100% ethanol were added and stirred, and then centrifuged to obtain a precipitate. The obtained precipitate was washed and air-dried, and then dissolved in water subjected to DEPC treatment to obtain an RNA solution.

Collection method 2 (with centrifuge treatment and without nonwoven fabric filter treatment):
To the frozen stool, a phenol mixture “Trizol” (manufactured by Invitrogen) was added and dissolved sufficiently, followed by centrifugation at 12,000 × g and 4 ° C. for 5 minutes. Chloroform was added to the supernatant (aqueous phase) obtained by the centrifugation treatment and mixed well using a vortex. Thereafter, centrifugation was performed at 12,000 × g and 4 ° C. for 20 minutes. RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. The ethanol precipitation method was performed in the same manner as in the recovery method 1.

Collection method 3 (when there is no centrifugal separation treatment and with non-woven fabric filter treatment):
To the frozen stool, a phenol mixture “Trizol” (manufactured by Invitrogen) was added and dissolved sufficiently to prepare a suspension. This suspension was placed in a filter-equipped Sanispec test bag (manufactured by ASONE), passed through a nonwoven fabric filter, and the obtained filtrate was collected in a 15 mL tube. Furthermore, after adding chloroform and mixing well using a vortex, it centrifuged at 12,000 * g and 4 degreeC for 20 minute (s). RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. The ethanol precipitation method was performed in the same manner as in the recovery method 1.

Collection method 4 (in the case of centrifugal separation treatment and nonwoven fabric filter treatment):
To the frozen stool, a phenol mixture “Trizol” (manufactured by Invitrogen) was added and dissolved sufficiently, followed by centrifugation at 12,000 × g and 4 ° C. for 5 minutes. The supernatant (aqueous phase) obtained by the centrifugation was placed in a filter-equipped Sanispec test bag (manufactured by ASONE), passed through a nonwoven fabric filter, and the resulting filtrate was collected in a 15 mL tube. Chloroform was added to the filtrate and mixed well using a vortex, and then centrifuged at 12,000 × g and 4 ° C. for 20 minutes. RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. The ethanol precipitation method was performed in the same manner as in the recovery method 1.

The xenoRNA in the recovered RNA was analyzed.
Specifically, for each reaction system, 4.0 μL or 1.0 μL of RNA solution recovered from stool and 1 μL of xenoRNA were added, and a reverse transcription reaction was performed to obtain cDNA. As a control, only 1 μL of xenoRNA was added to perform a reverse transcription reaction.
Subsequently, Taqman PCR was used for amplification using the xenoRNA Taqman probe, and the resulting amplification product was detected. As a primer for real-time PCR, a probe (Cat: # 4386995) attached to the cell-to-PCR control kit manufactured by Applied Biosystems was used. Specifically, 1 μL of each cDNA was dispensed into a 0.2 mL 96-well PCR plate. Thereafter, 8 μL of ultrapure water and 10 μL of nucleic acid amplification reagent “TaqMan GeneExpression Master Mix” (Applied Biosystems) were added to each well, and 1 μL of probe was added and mixed, and the PCR reaction solution was mixed. Prepared. The PCR plate was placed in an ABI real-time PCR apparatus, treated at 95 ° C. for 10 minutes, and then subjected to 40 cycles of thermal cycling at 95 ° C. for 1 minute, 56.5 ° C. for 1 minute, and 72 ° C. for 1 minute. Further, PCR was performed while measuring the fluorescence intensity over time by treating at 72 ° C. for 7 minutes.

  Table 1 and FIG. 1 show the relative values of the expression level of xenoRNA when the measurement result of fluorescence intensity is analyzed and the expression level (copy number) of xenoRNA alone is 1. In the table, “centrifugation (−), non-woven fabric filter (−)” is the result of using the RNA solution recovered by the recovery method 1, and “centrifugation (+), non-woven fabric filter (−)” is the recovery method 2. The results of using the recovered RNA solution are the results of using the RNA solution recovered by the recovery method 3 for “centrifugation (−), non-woven filter (+)” and “centrifuging (+), non-woven filter ( “+)” Indicates the results obtained using the RNA solution recovered by the recovery method 4, respectively. “Control” shows the results when no RNA derived from E. coli was added. In the column “RT template”, RNA added as a template to the reverse transcription reaction is shown.

  For RNA collected from stool collected from healthy person B, the expression level of xenoRNA is the same regardless of the amount of RNA solution added to the reverse transcription reaction and the subsequent PCR reaction, and the presence or absence of centrifugation or non-woven fabric filter. There was no big difference. On the other hand, RNA recovered from stool collected from a healthy person A is recovered by a recovery method 2 using centrifugation, a recovery method 3 using a nonwoven fabric filter, and a recovery method 4 using a centrifugation and a nonwoven fabric filter. In the RNA thus obtained, the same xenoRNA expression level was observed regardless of the amount of RNA solution added to the reverse transcription reaction. On the other hand, in the RNA recovered by the recovery method 1 without using any of the centrifugation treatment and the nonwoven fabric filter, the expression level of xenoRNA is significantly lower than the RNA recovered by the recovery methods 2 to 4, in particular, When 4 μL of RNA solution was added to the reverse transcription reaction, there was almost no xenoRNA expression level.

  All samples contained the same amount of xenoRNA, but when RNA derived from feces A was added, the expression level of xenoRNA was greatly different depending on the recovery method and the amount of RNA added. This is presumably because the amount of stool-derived inhibitory substance brought into each reaction solution is different. Here, it can be said that the smaller the expression level of xenoRNA, the greater the inhibitory effect of the stool-derived inhibitory substance. That is, from these results, the suspension of the inhibitor is carried over by centrifuging the suspension, bringing the supernatant after the centrifuging into contact with the nonwoven fabric, and bringing the suspension into contact with the nonwoven fabric. It is clear that low purity nucleic acids can be recovered. In particular, the expression of xenoRNA was higher when the RNA recovered by the recovery method 4 was added than when the RNA recovered by the recovery method 2 or 3 was added. It was found that a very high inhibitory substance removal effect can be obtained by combining the separation treatment and the nonwoven fabric filter treatment. In addition, feces collected from a healthy person B in which the effects of the recovery method and the amount of RNA added were hardly observed are relatively low in impurities such as inhibitors, and a relatively large amount of feces collected from a healthy person A is contained. It is guessed that it was.

[Example 2]
Nucleic acids were collected by the collection method of the present invention using a bacterial group isolated and cultured from feces as a biological sample.
First, stool collected from one healthy person was suspended in physiological saline, and a part of the suspension was separated and cultured on a plate medium to obtain a bacterial group. This bacterial group is cultured in a liquid medium, and the obtained culture solution is put into 12 15 mL tubes each 10 mL. After centrifugation, the supernatant is discarded, and only the pelleted bacteria are frozen in liquid nitrogen. Stored at 80 ° C. According to the recovery methods 1 to 4 of Example 1, RNA solutions were prepared from bacterial cultures contained in each of three 15 mL tubes.
The xenoRNA in the recovered RNA was analyzed. Specifically, in the same manner as in Example 1, for each reaction system, 4.0 μL or 1.0 μL of RNA solution recovered from stool and 1 μL of xenoRNA were added to perform a reverse transcription reaction, and the obtained cDNA was used as a template. As described above, PCR was performed while measuring the fluorescence intensity over time.

Table 2 and FIG. 2 show the relative value of the expression level of xenoRNA when the measurement result of fluorescence intensity is analyzed and the expression level (copy number) of xenoRNA alone is 1. In the table, “centrifugation (−), non-woven filter (−)” and the like are the same as those in Table 1.
As a result, in the same manner as in Example 1, when the suspension was centrifuged, the resulting supernatant (aqueous layer) was brought into contact with the nonwoven fabric. It was found that a highly pure nucleic acid with less inhibitor carryover can be recovered than in the case of the above.

[Example 3]
In the recovery method of the present invention, the influence of centrifugal force when centrifuging a suspension prepared from a biological sample and a cell lysing agent was observed.
First, stool collected from 3 healthy individuals was dispensed into six 15 mL tubes of approximately 0.5 g each, frozen in liquid nitrogen, and stored at −80 ° C. Subsequently, a phenol mixture “Trizol” (manufactured by Invitrogen) was added to each frozen stool contained in each 15 mL tube and dissolved sufficiently to prepare a suspension.
Three of six 15 mL tubes were centrifuged at 2000 × g and 4 ° C. for 5 minutes. The remaining three were centrifuged at 12000 × g and 4 ° C. for 5 minutes. Then, after adding chloroform and mixing well using a vortex, it centrifuged at 12,000g and 4 degreeC for 20 minute (s). RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. The ethanol precipitation method was performed in the same manner as in the recovery method 1. The concentration of each recovered RNA was measured. The measurement results are shown in Table 3 and FIG.

The expression level of β2M gene in the recovered RNA was analyzed.
Specifically, first, 1.0 μL of RNA solution recovered from stool was added per reaction system, and reverse transcription reaction was performed to obtain cDNA.
Next, Taqman PCR was performed using the β2M Taqman probe to amplify, and the resulting amplification product was detected. As a primer for real-time PCR, β2M primer probe MIX (Catalog No. 4326319E) manufactured by Applied Biosystems was used. Specifically, 4 μL of each cDNA was dispensed into a 0.2 mL 96-well PCR plate. Thereafter, 8 μL of ultrapure water and 10 μL of nucleic acid amplification reagent “TaqMan GeneExpression Master Mix” (Applied Biosystems) were added to each well, and 1 μL of probe was added and mixed, and the PCR reaction solution was mixed. Prepared. The PCR plate was placed in an ABI real-time PCR apparatus, treated at 95 ° C. for 10 minutes, and then subjected to 40 cycles of thermal cycling at 95 ° C. for 1 minute, 56.5 ° C. for 1 minute, and 72 ° C. for 1 minute. Further, PCR was performed while measuring the fluorescence intensity over time by treating at 72 ° C. for 7 minutes. The expression level of β2M gene (measured value of fluorescence intensity) is shown in Table 3 and FIG.

  As a result, the stool specimen and the cell lysing agent were mixed, and the prepared suspension was centrifuged at 12000 × g or 2000 × g. As a result, in each stool specimen, the RNA concentration and β2M gene expression level were There was no significant difference between them.

[Example 4]
In the recovery method of the present invention, the influence of the type of fiber brought into contact with the liquid fraction containing nucleic acid was observed.
First, stool collected from one healthy person was collected into 33 15 mL tubes of about 0.5 g each, snap-frozen using liquid nitrogen, and stored at −80 ° C.
Subsequently, a phenol mixture “Trizol” (manufactured by Invitrogen) was added to each of the frozen stool contained in 33 15 mL tubes and dissolved sufficiently to prepare a suspension. For each of three of the 15 15 mL tubes, the carrier made of the fibers shown in Table 4 was added to the suspension contained in each tube, and vortexed for 30 seconds. Then, after adding chloroform and mixing well using a vortex, it centrifuged at 12,000 xg and 4 degreeC for 20 minute (s). RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. Specifically, sodium acetate and 100% ethanol were added and stirred, and then centrifuged to obtain a precipitate. The obtained precipitate was washed and air-dried, and then dissolved in water subjected to DEPC treatment to obtain an RNA solution.
In addition, an RNA solution was prepared from the suspension in the remaining three 15 mL tubes by the recovery method 1 of Example 1 (when neither centrifugation nor nonwoven fabric filter treatment was used).

The xenoRNA in the recovered RNA was analyzed.
Specifically, for each reaction system, 4.0 μL or 1.0 μL of RNA solution recovered from stool and 1 μL of xenoRNA were added, and a reverse transcription reaction was performed to obtain cDNA. As a control, only 1 μL of xenoRNA was added to perform a reverse transcription reaction.
Next, PCR was performed using the obtained cDNA as a template in the same manner as in Example 1 while measuring the fluorescence intensity over time.

Table 5 and FIG. 5 show the relative values of the expression level of xenoRNA when the measurement result of fluorescence intensity is analyzed and the expression level (copy number) of xenoRNA alone is 1. In Table 5, “Control 1” is the result when RNA recovered by the recovery method 1 of Example 1 was used as a template for RT, and “Control 2” used only 1 μL of xenoRNA as a template for RT. Is the result of the case.
As a result, when silica compound fibers such as silica and glass were used (fibers 1, 2 and 10), xenoRNA expression was observed when 1 μL and 4 μL of the RNA solution recovered from stool was used as the RT template. The difference in amount was large and there was a lot of inhibitor carryover.
On the other hand, when other chemical fibers are used except the fiber 5, 1 μL or 4 μL of the RNA solution recovered from the stool is used as the RT template regardless of the fiber form. Thus, it was found that the xenoRNA expression level difference was small and carryover of the inhibitor was suppressed.

  When filter paper made of cellulose fiber is used (fiber 5), it is slightly better than the case of silica compound fiber such as fiber 1, but unlike non-woven fabric made of cellulose fiber (fiber 9), feces The difference in the xenoRNA expression level was great when 1 μL and 4 μL of the RNA solution recovered from the sample was used, and the influence of carryover of the inhibitor was observed. This is presumably due to the difference in shape between the filter paper and the nonwoven fabric, particularly the difference in pore diameter. The pore diameter of the filter paper is about 0.45 μm, which is smaller than that of the nonwoven fabric. For this reason, the suspension penetrates sufficiently into the nonwoven fabric and the inhibitory substances are easily adsorbed to the nonwoven fabric, but the suspension does not easily penetrate into the filter paper having a small pore size, and the adsorption of the inhibitory substances becomes insufficient. It is assumed that it is easy.

[Example 5]
After collecting feces from 96 healthy individuals, about 0.5 g each was taken into four 15 mL tubes as soon as possible, snap-frozen using liquid nitrogen, and stored at −80 ° C. An RNA solution was prepared by the recovery methods 1 to 4 in Example 1 for each stool contained in a 15 mL tube collected from each healthy person.
Subsequently, the β2M gene expression level in the recovered RNA was analyzed. Specifically, in the same manner as in Example 3, cDNA was obtained from an RNA solution collected from stool, amplified using Taqman PCR using the β2M Taqman probe with the cDNA as a template, and the obtained amplification product was obtained. Detected. The measurement results of the fluorescence intensity are analyzed, and the expression level of β2M gene (measurement value of fluorescence intensity) is shown in Tables 6-8. In addition, “◯” was added to the left of the result of the recovery method (fluorescence intensity) in which the expression level of the β2M gene was the highest in each specimen. “Centrifuge separation (−), nonwoven fabric filter (−)” and the like in each table are the same as those in Table 1.

As a result, 2 samples out of 96 samples showed a low expression level in any recovery method, probably because the amount of contained cells was small. In particular, when the nonwoven fabric filter treatment was performed alone (collection method 3), the expression level was less than the detection limit (“UND” in the table).
In addition, after the centrifugal separation treatment, the result of the non-woven fabric filter treatment (recovery method 4), the case where neither the centrifugal separation treatment nor the non-woven fabric filter treatment is performed (recovery method 1), or the case of performing either alone (recovery method 2 or 3) Compared to the above, 64 specimens (67%) out of 96 specimens showed a high expression level. Thus, it was revealed that the method using both treatments had the highest inhibitory substance removal effect. In addition, 15 samples (16%) showed the highest expression in the case of only the centrifugal separation treatment, and 14 samples (15%) showed the highest expression in the case of only the untreated cloth treatment. ), 31% in total. In the case of the collection method 4 in which both treatments were used in combination, there was no specimen in which the expression level was extremely lower than in the other methods.
From these results, it became clear that a high inhibitory substance removal effect can be obtained for a larger number of biological samples by using the centrifugal separation process and the unemployed cloth filter process in combination.

  Since the nucleic acid with high purity can be recovered from a biological sample such as stool by the recovery method of the present invention, it can be used particularly in the field of clinical examination such as periodic medical examination using the biological sample.

Claims (13)

A method for recovering nucleic acid from a biological sample, comprising:
(A) mixing a biological sample and a cell lysing agent to prepare a suspension;
(B) centrifuging the suspension prepared in the step (A) and recovering the supernatant as a crude nucleic acid solution;
(C) recovering nucleic acid from the crude nucleic acid solution recovered in the step (B),
Have
A treatment in which chemical fiber or natural fiber (excluding silica compound fiber) is contacted,
Before the step (B), with respect to the suspension prepared by the step (A),
The nucleic acid recovery, which is performed on the crude nucleic acid solution recovered in the step (B) before the step (C) or on the recovered nucleic acid after the step (C). Method.   2. The method for recovering nucleic acid according to claim 1, wherein the chemical fiber or the natural fiber forms a porous body.   The method for recovering nucleic acid according to claim 2, wherein the chemical fiber or the natural fiber has an average pore diameter of 1 to 600 µm.   The method for recovering nucleic acid according to any one of claims 1 to 3, wherein the chemical fiber or natural fiber is a non-woven fabric.   The nucleic acid recovery method according to any one of claims 1 to 4, wherein the suspension contains an RNase inhibitor.   The method for recovering nucleic acid according to any one of claims 1 to 5, wherein the centrifugal force of the centrifugation treatment is 2000 xg or more.   The nucleic acid recovery method according to any one of claims 1 to 6, wherein the biological sample is feces. A method for analyzing nucleic acids in a biological sample,
(A) mixing a biological sample and a cell lysing agent to prepare a suspension;
(B) centrifuging the suspension prepared in the step (A) and recovering the supernatant as a crude nucleic acid solution;
(C) recovering nucleic acid from the crude nucleic acid solution recovered in the step (B),
(D) analyzing the nucleic acid recovered in the step (C);
Have
A treatment in which chemical fiber or natural fiber (excluding silica compound fiber) is contacted,
Before the step (B), with respect to the suspension prepared by the step (A),
Nucleic acid analysis, which is performed on the crude nucleic acid solution recovered in the step (B) before the step (C) or on the recovered nucleic acid after the step (C). Method.   The nucleic acid analysis method according to claim 8, wherein in the step (D), a nucleic acid derived from a mammalian cell is analyzed.   The nucleic acid-derived nucleic acid analysis method according to claim 9, wherein the mammalian cell-derived nucleic acid is a marker indicating neoplastic conversion or a marker indicating inflammatory digestive tract disease.   In the step (D), a reverse transcription reaction is carried out using the RNA recovered in the step (C) as a template, and using the obtained cDNA, a marker indicating neoplastic transformation or a marker indicating inflammatory digestive organ disease The method for analyzing a nucleic acid according to claim 8 or 9, wherein: A kit used for recovering nucleic acid from a biological sample,
A nucleic acid recovery kit comprising a cell lysing agent and chemical fibers or natural fibers (excluding silica compound fibers).   The kit for nucleic acid recovery according to claim 12, wherein the chemical fiber or natural fiber is a nonwoven fabric.

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