JP2018038352A - Nucleic acid dissolution adsorption reagent, nucleic acid extraction reagent set, nucleic acid extraction kit, and nucleic acid extraction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid dissolution adsorption reagent that can extract a nucleic acid efficiently.SOLUTION: A nucleic acid dissolution adsorption reagent contains a chaotropic agent and an additive. The additive contains at least one of albumin, skim milk, gelatin, casein, and globulin. The reagent dissolves a nucleic acid and allows the dissolved nucleic acid to be adsorbed on a nucleic acid-bonding solid phase carrier whose surface is a silicon oxide layer.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a nucleic acid dissolution and adsorption reagent, a nucleic acid extraction reagent set, a nucleic acid extraction kit, and a nucleic acid extraction method.

  A polymerase chain reaction (PCR) is known as a method for extracting nucleic acids such as bacteria and viruses and specifically amplifying and detecting the nucleic acids. The PCR method is widely used for genetic testing. However, when the PCR method is routinely used in clinical practice, PCR is inhibited by the influence of components contained in the specimen, and thus a process for extracting and purifying and recovering nucleic acids by removing the inhibitor from the specimen is required. .

  For example, Patent Document 1 describes a method of extracting a nucleic acid from a biological material (such as whole blood) by binding the nucleic acid to a carrier having a silica surface in the presence of a chaotropic salt.

Japanese Patent Application Laid-Open No. 11-146783

  However, for example, in the diagnosis of infectious diseases typified by influenza in the medical field, in order to confirm infection at an earlier stage (stage where the amount of bacteria and viruses is small) after the disease, Is required to be extracted efficiently.

  One of the objects according to some embodiments of the present invention is to provide a nucleic acid dissolution and adsorption reagent that can efficiently extract a nucleic acid. Moreover, one of the objects according to some aspects of the present invention is to provide a nucleic acid extraction reagent set including the nucleic acid dissolution and adsorption reagent. Another object of some embodiments of the present invention is to provide a nucleic acid extraction kit including a container in which the nucleic acid dissolution and adsorption reagent is accommodated. Another object of some aspects of the present invention is to provide a nucleic acid extraction method that can efficiently extract nucleic acids.

The nucleic acid dissolution and adsorption reagent according to the present invention comprises:
A chaotropic agent and an additive,
The additive includes at least one of albumin, skim milk, gelatin, casein, and globulin,
The nucleic acid is dissolved, and the dissolved nucleic acid is adsorbed on a nucleic acid-binding solid phase carrier whose surface is silicon oxide.

  With such a nucleic acid dissolution and adsorption reagent, nucleic acids can be extracted efficiently (for details, refer to “3. Experimental Examples” described later).

In the nucleic acid dissolution and adsorption reagent according to the present invention,
The additive may be bovine serum albumin.

With such a nucleic acid dissolution and adsorption reagent, nucleic acids can be extracted more reliably and efficiently (for details, see “3. Experimental Examples” described later).

In the nucleic acid dissolution and adsorption reagent according to the present invention,
The concentration of the additive contained in the nucleic acid dissolution / adsorption reagent may be 0.001 mg / mL or more and 15 mg / mL or less.

  With such a nucleic acid dissolution and adsorption reagent, nucleic acid can be extracted more efficiently (for details, refer to “3. Experimental Example” described later).

In the nucleic acid dissolution and adsorption reagent according to the present invention,
The concentration of the additive contained in the nucleic acid dissolution / adsorption reagent may be 0.1 mg / mL or more and 1.5 mg / mL or less.

  With such a nucleic acid dissolution and adsorption reagent, nucleic acid can be extracted more efficiently (for details, refer to “3. Experimental Example” described later).

The nucleic acid extraction reagent set according to the present invention comprises:
A nucleic acid dissolution and adsorption reagent according to the present invention;
An acidic nucleic acid washing reagent for washing the nucleic acid-binding solid phase carrier adsorbed with nucleic acid;
A nucleic acid elution reagent for separating and eluting nucleic acid from the washed nucleic acid-binding solid phase carrier;
including.

  Since such a nucleic acid extraction reagent set includes the nucleic acid dissolution and adsorption reagent according to the present invention, nucleic acid can be extracted efficiently.

In the nucleic acid extraction reagent set according to the present invention,
The pH of the nucleic acid washing reagent may be less than 4.7.

  With such a nucleic acid extraction reagent set, nucleic acids can be extracted more reliably and efficiently (for details, see “3. Experimental Examples” described later).

The nucleic acid extraction kit according to the present invention comprises:
A first container containing a nucleic acid dissolving and adsorbing reagent according to the present invention;
A second container containing an acidic nucleic acid washing reagent for washing the nucleic acid binding solid phase carrier to which the nucleic acid has been adsorbed;
A third container containing a nucleic acid elution reagent for separating and eluting nucleic acid from the washed nucleic acid binding solid phase carrier;
including.

  Since such a nucleic acid extraction reagent set includes a container in which the nucleic acid dissolution and adsorption reagent according to the present invention is contained, nucleic acid can be extracted efficiently.

The nucleic acid extraction method according to the present invention comprises:
Adsorbing nucleic acid on a nucleic acid-binding solid phase carrier whose surface is silicon oxide in a nucleic acid dissolution and adsorption reagent containing a chaotropic agent and an additive;
Placing the nucleic acid binding solid phase carrier adsorbed with nucleic acid in an acidic nucleic acid washing reagent, and washing the nucleic acid binding solid phase carrier adsorbed with nucleic acid;
After the washing step, placing the nucleic acid-binding solid phase carrier adsorbed with nucleic acid in a nucleic acid elution reagent, and eluting the nucleic acid;
Including
The additive includes at least one of albumin, skim milk, gelatin, casein, and globulin.

  In such a nucleic acid extraction method, nucleic acid can be extracted efficiently.

Sectional drawing which shows typically the nucleic acid extraction kit which concerns on this embodiment. The flowchart for demonstrating the nucleic acid extraction method which concerns on this embodiment. The graph which shows Ct value with respect to the presence or absence of bovine serum albumin. The graph which shows Ct value with respect to the presence or absence of bovine serum albumin. The graph which shows Ct value with respect to the addition amount of a bovine serum albumin. The graph which shows the number of copies in each process. The graph for demonstrating the relationship between the pH of a silica, bovine serum albumin, and a nucleic acid, and an electric charge. Sectional drawing which shows typically the state which the bovine serum albumin adsorb | sucked to the nucleic acid binding solid-phase carrier.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.

1. Nucleic Acid Extraction Kit First, the nucleic acid extraction kit according to this embodiment will be described with reference to the drawings. FIG. 1 is a cross-sectional view schematically showing a nucleic acid extraction kit 100 according to this embodiment.

  The nucleic acid extraction kit 100 includes a nucleic acid extraction reagent set according to the present invention (in the illustrated example, the nucleic acid extraction reagent set 10). The nucleic acid extraction reagent set 10 is a reagent set for extracting nucleic acids. The nucleic acid extraction reagent set 10 includes a nucleic acid lysis adsorption reagent (nucleic acid lysis adsorption reagent 11 in the illustrated example), a first nucleic acid washing reagent 12, a second nucleic acid washing reagent 13, and a third nucleic acid washing reagent 14 according to the present invention. And a nucleic acid elution reagent 15. Further, the nucleic acid extraction kit 100 includes a nucleic acid-binding solid phase carrier 20, containers 30, 31, 32, 33, 34, and 35, and a housing 40. The nucleic acid to be extracted (target nucleic acid, nucleic acid contained in the specimen) may be DNA (deoxyribonucleic acid) or RNA (ribonucleic acid).

1.1. Nucleic acid dissolution and adsorption reagent The nucleic acid dissolution and adsorption reagent 11 is a reagent for dissolving nucleic acid and adsorbing the dissolved nucleic acid to the nucleic acid binding solid phase carrier 20. The nucleic acid dissolution adsorption reagent 11 is stored in a container 30 in a liquid state. The nucleic acid dissolution adsorption reagent 11 is, for example, a nucleic acid dissolution adsorption solution. The nucleic acid dissolution adsorption reagent 11 includes, for example, a chaotropic agent, a surfactant, a buffer, an additive, and a solvent.

A chaotropic agent is a substance having the action of generating chaotropic ions in an aqueous solution and increasing the water solubility of hydrophobic molecules. The presence of the chaotropic agent in the aqueous solution makes it more thermodynamically stable when the nucleic acid in the aqueous solution is adsorbed to a solid than when surrounded by water. Adsorb to 20 surfaces. Examples of the chaotropic agent include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, sodium perchlorate and the like. Since guanidine thiocyanate has a particularly large protein denaturing action, it can be suitably used as a chaotropic agent. The concentration of the chaotropic agent contained in the nucleic acid dissolution adsorption reagent 11 is, for example, 40% by mass or more and 80% by mass or less.

  The surfactant has a function of destroying the cell membrane. Furthermore, the surfactant has a function of denaturing proteins contained in the cells. Examples of the surfactant include a triton surfactant such as Triton X-100, a nonionic surfactant such as a tween surfactant such as Tween 20, and an anion such as sodium N-uraroyl sarcosine (SDS). Surfactants and the like. The concentration of the surfactant contained in the nucleic acid dissolution / adsorption reagent 11 is, for example, 0.5% by mass or more and 5% by mass or less.

  The buffer has a function of adjusting the pH of the nucleic acid dissolution adsorption reagent 11. The pH of the nucleic acid dissolution adsorption reagent 11 is, for example, 6 or more and 8 or less. The buffer is, for example, trishydroxymethylaminomethane (Tris (hydroxymethyl) aminomethane). The concentration of the buffer contained in the nucleic acid dissolution / adsorption reagent 11 is, for example, 0.1% by mass or more and 1% by mass or less.

  The additive has a function of efficiently extracting a nucleic acid. The additive includes at least one of albumin, skim milk, gelatin, casein, and globulin. Preferably, the additive is bovine serum albumin (BSA). Bovine serum albumin is a protein purified from bovine serum. The concentration of the additive contained in the nucleic acid dissolution adsorption reagent 11 is, for example, 0.001 mg / mL or more and 15 mg / mL or less, preferably 0.01 mg / mL or more and 15 mg / mL or less, more preferably 0.1 mg. / ML or more and 15 mg / mL or less, and still more preferably 0.1 mg / mL or more and 1.5 mg / mL or less. By setting the concentration of the additive within the above range, nucleic acids can be extracted more efficiently.

  The solvent is, for example, water. The solvent may further contain alcohol (ethanol). When the solvent contains ethanol, for example, the nucleic acid dissolution and adsorption reagent 11 adds 30 v / v% or more and 70 v / v% or less of ethanol to an aqueous solution containing a chaotropic agent, a surfactant, a buffer, and an additive. Prepared.

1.2. First Nucleic Acid Washing Reagent The first nucleic acid washing reagent 12 is a reagent for washing the nucleic acid binding solid phase carrier 20 to which the nucleic acid has been adsorbed. The first nucleic acid washing reagent 12 is stored in a container 31 in a liquid state. The first nucleic acid washing reagent 12 is, for example, a first nucleic acid washing solution. The first nucleic acid washing reagent 12 includes, for example, a chaotropic agent and a solvent.

  As the chaotropic agent, for example, those mentioned in “1.1. Nucleic acid dissolution and adsorption reagent” are used. For example, when guanidine hydrochloride is used as the chaotropic agent, the nucleic acid-binding solid phase carrier 20 can be washed while maintaining or enhancing the adsorption of the nucleic acid adsorbed on the nucleic acid-binding solid phase carrier 20. The concentration of the chaotropic agent contained in the first nucleic acid washing reagent 12 is, for example, 40% by mass or more and 80% by mass or less.

  The solvent is, for example, water. The solvent may further contain ethanol. When the solvent contains ethanol, the first nucleic acid washing reagent 12 is prepared, for example, by adding 30 v / v% or more and 70 v / v% or less of ethanol to an aqueous solution containing a chaotropic agent.

  The first nucleic acid washing reagent 12 may contain the surfactant mentioned in “1.1. Nucleic acid dissolution and adsorption reagent”. The pH of the first nucleic acid washing reagent 12 is, for example, 6 or more and 8 or less.

1.3. Second nucleic acid washing reagent The second nucleic acid washing reagent 13 is a reagent for washing the nucleic acid binding solid phase carrier 20 to which the nucleic acid has been adsorbed. For example, the nucleic acid binding solid phase washed by the first nucleic acid washing reagent 12 is used. This is a reagent for further washing the phase carrier 20. The second nucleic acid washing reagent 13 is housed in the container 32 in a liquid state. The second nucleic acid cleaning reagent 13 is, for example, a second nucleic acid cleaning liquid. The second nucleic acid washing reagent 13 is, for example, ethanol. Ethanol can suppress the introduction of a chaotropic agent into the nucleic acid elution reagent 15. A chaotropic agent causes PCR inhibition when PCR is performed on the extracted nucleic acid.

  The second nucleic acid washing reagent 13 may further contain a buffer such as Tris and a salt such as NaCl. When the second nucleic acid washing reagent 13 contains a buffer and a salt, the second nucleic acid washing reagent 13 is an aqueous solution containing, for example, 50 v / v% or more and 90 v / v% or less of the buffer and salt with respect to ethanol. It is prepared by adding. The concentration of the buffer contained in the aqueous solution is, for example, from 0.1% by mass to 1% by mass, and the concentration of the salt contained in the aqueous solution is, for example, from 0.3% by mass to 3% by mass.

  The second nucleic acid washing reagent 13 may contain a chaotropic agent, but in order to eliminate the introduction of the chaotropic agent into the third nucleic acid washing reagent 14 and the nucleic acid elution reagent 15, the second nucleic acid washing reagent 13 is It is preferable that no chaotropic agent is contained. The pH of the second nucleic acid washing reagent 13 is, for example, 6 or more and 8 or less.

1.4. Third nucleic acid washing reagent The third nucleic acid washing reagent 14 is a reagent for washing the nucleic acid binding solid phase carrier 20 to which the nucleic acid has been adsorbed. For example, the nucleic acid binding solid phase washed by the second nucleic acid washing reagent 13 is used. This is a reagent for further washing the phase carrier 20. The third nucleic acid washing reagent 14 is accommodated in the container 33 in a liquid state. The third nucleic acid washing reagent 14 is, for example, a third nucleic acid washing solution. The third nucleic acid washing reagent 14 includes, for example, a buffer and a solvent.

  The buffer has a function of adjusting the pH of the third nucleic acid washing reagent 14. The third nucleic acid washing reagent is acidic. The pH of the third nucleic acid washing reagent 14 is, for example, less than 4.7, and preferably 2 or more and 4.5 or less. The buffer is not particularly limited as long as the pH of the third nucleic acid washing reagent 14 can be made less than 4.7, but for example, citric acid or the like. The concentration of the buffer contained in the third nucleic acid washing reagent 14 is, for example, 1 mM or more and 10 mM or less.

  The solvent is, for example, water and does not contain alcohol such as ethanol. Thereby, it is possible to suppress ethanol from being brought into the nucleic acid elution reagent 15. Further, the buffer such as citric acid has a function of suppressing the introduction of ethanol into the nucleic acid elution reagent 15. Ethanol causes PCR inhibition when PCR is performed on the extracted nucleic acid.

  The third nucleic acid washing reagent 14 may contain the surfactant mentioned in “1.1. Nucleic acid dissolution and adsorption reagent”. When the third nucleic acid washing reagent 14 contains a surfactant, the third nucleic acid washing reagent 14 contains 0.01 v / v% or more and 0.1 v / v% or less of the surfactant with respect to the aqueous solution containing the buffer. It is prepared by adding an aqueous solution containing it.

1.5. Nucleic acid elution reagent The nucleic acid elution reagent 15 is a reagent for separating (desorbing) and eluting nucleic acid from the washed nucleic acid-binding solid phase carrier 20. The nucleic acid elution reagent 15 is contained in a container 34 in a liquid state. The nucleic acid elution reagent 15 is, for example, a nucleic acid eluate. The nucleic acid elution reagent 15 is, for example, water (for example, sterilized water obtained by boiling and disinfecting water). Nucleic acid elution reagent 15 is Tris-HCl
(For example, 5 mM, pH 8.5) etc. may be included. The pH of the nucleic acid elution reagent 15 is, for example, 6 or more and 9 or less, preferably weakly alkaline, and optimally pH 8.5.

1.6. Nucleic acid-binding solid phase carrier The nucleic acid-binding solid phase carrier 20 is a substance capable of adsorbing a nucleic acid, that is, holding it by reversible physical adsorption. The nucleic acid binding solid phase carrier 20 is accommodated in the container 35 together with the liquid 22. Although the liquid 22 is not specifically limited, For example, it is water. The liquid 22 may contain a salt such as sodium chloride or lithium chloride. Thereby, the specific gravity of the liquid 22 can be adjusted, and sedimentation of the magnetic beads can be prevented (the sedimentation speed of the magnetic beads can be reduced). Although not shown, the nucleic acid-binding solid phase carrier 20 may be housed alone in the container 35 instead of in the liquid 22.

  The nucleic acid-binding solid phase carrier 20 has a fine particle shape, for example. A plurality of nucleic acid-binding solid phase carriers 20 are provided in the container 35. The number of the nucleic acid binding solid phase carriers 20 is not particularly limited. For example, the nucleic acid-binding solid phase carrier 20 preferably has magnetism in order to move the inside of the container in a desired direction while adsorbing the nucleic acid. The nucleic acid-binding solid phase carrier 20 is, for example, a magnetic bead whose surface is silicon oxide. Specifically, the nucleic acid-binding solid phase carrier 20 is a magnetic bead in which a silicon dioxide (silica) layer is coated on the surface of a metal particle.

1.7. Container The containers 30, 31, 32, 33, 34, and 35 contain the nucleic acid dissolution and adsorption reagent 11, the nucleic acid washing reagents 12, 13, and 14, the nucleic acid elution reagent 15, and the nucleic acid-binding solid phase carrier 20, respectively. . Each of the containers 30, 31, 32, 33, 34, and 35 has a container main body and a lid that engages with the container main body. The shape and material of the containers 30, 31, 32, 33, 34, and 35 are not particularly limited as long as the contents can be accommodated and sealed.

1.8. Case The case 40 accommodates the containers 30, 31, 32, 33, 34, and 35. If the housing | casing 40 can accommodate the containers 30, 31, 32, 33, 34, and 35, the shape and material will not be specifically limited.

  The nucleic acid dissolution adsorption reagent 11 has the following characteristics, for example.

  The nucleic acid dissolution adsorption reagent 11 includes a chaotropic agent and an additive, and the additive includes at least one of albumin, skim milk, gelatin, casein, and globulin. Therefore, the nucleic acid dissolution adsorption reagent 11 can efficiently extract nucleic acid (for details, refer to “3. Experimental example” described later). This makes it possible to extract and collect a smaller amount of nucleic acid in a specimen. For example, an infectious disease is detected at the initial stage of virus amplification and treatment is started at the initial stage. Can be prevented. Even when only a small amount of sample can be collected, a highly accurate test can be performed.

  In the nucleic acid dissolution adsorption reagent 11, the additive is bovine serum albumin. Therefore, the nucleic acid dissolution adsorption reagent 11 can extract the nucleic acid more reliably and efficiently (for details, refer to “3. Experimental example” described later).

  The nucleic acid dissolution adsorption reagent 11 contains 0.001 mg / mL or more and 15 mg / mL or less additive, and more preferably 0.1 mg / mL or more and 1.5 mg / mL or less. Therefore, the nucleic acid dissolution adsorption reagent 11 can extract nucleic acid more efficiently (for details, refer to “3. Experimental example” described later).

  The nucleic acid extraction reagent set 10 includes a nucleic acid dissolution and adsorption reagent 11, an acidic third nucleic acid washing reagent 14, and a nucleic acid elution reagent 15. Therefore, the nucleic acid extraction reagent set 10 can efficiently extract nucleic acids.

  In the nucleic acid extraction reagent set 10, the pH of the third nucleic acid washing reagent 14 is less than 4.7. Therefore, the nucleic acid extraction reagent set 10 can extract nucleic acids more reliably and efficiently (for details, refer to “3. Experimental example” described later).

  The nucleic acid extraction kit 100 includes a container 30 in which the nucleic acid dissolution and adsorption reagent 11 is stored, a container 33 in which the acidic third nucleic acid washing reagent 14 is stored, and a container 34 in which the nucleic acid elution reagent 15 is stored. Therefore, the nucleic acid extraction kit 100 can efficiently extract nucleic acids.

  In the above example, an example including three (three types) of nucleic acid washing reagents 12, 13, and 14 has been described. However, the nucleic acid extraction reagent set and the nucleic acid extraction kit according to the present invention include the third nucleic acid washing reagent 14. If it contains, although not shown in figure, it does not need to contain any one or both of the nucleic acid washing | cleaning reagents 12 and 13. However, by including three nucleic acid washing reagents, it is possible to purify the nucleic acid with high purity as compared with the case of containing two or less nucleic acid washing reagents. Although not shown, the nucleic acid extraction reagent set and the nucleic acid extraction kit according to the present invention may contain four or more nucleic acid washing reagents.

2. Nucleic Acid Extraction Method Next, a nucleic acid extraction method according to this embodiment will be described with reference to the drawings. FIG. 2 is a flowchart for explaining the nucleic acid extraction method according to this embodiment. Below, the nucleic acid extraction method using the nucleic acid extraction kit 100 mentioned above is demonstrated as an example.

  First, in the nucleic acid dissolution adsorption reagent 11, the nucleic acid is dissolved, and the dissolved nucleic acid is adsorbed to the nucleic acid binding solid phase carrier 20 (step S1, dissolution adsorption step). Specifically, the nucleic acid dissolution and adsorption reagent 11 is taken out from the container 30 and transferred to a container (not shown) (hereinafter referred to as “first extraction container”). Next, a sample (sample solution) containing a nucleic acid is introduced into the first extraction container and stirred. Thereby, the nucleic acid can be dissolved in the nucleic acid dissolution and adsorption reagent 11. Next, the nucleic acid-binding solid phase carrier 20 is taken out from the container 35, introduced into the first extraction container, and stirred. As a result, the dissolved nucleic acid can be adsorbed to the nucleic acid-binding solid phase carrier 20. Next, for example, the magnet is brought close to the side wall of the first extraction container, and the supernatant is discarded so that the nucleic acid-binding solid phase carrier 20 does not spill from the first extraction container.

  Next, the nucleic acid-binding solid phase carrier 20 on which the nucleic acid has been adsorbed is placed in the first nucleic acid washing reagent 12, and the nucleic acid-binding solid phase carrier 20 on which the nucleic acid has been adsorbed is washed (step S2, first washing step). Specifically, the first nucleic acid washing reagent 12 is taken out from the container 31 and introduced into the first extraction container, and the nucleic acid-binding solid phase carrier 20 is placed in the first nucleic acid washing reagent 12 and then stirred. Thereby, the nucleic acid-binding solid phase carrier 20 can be washed with the first nucleic acid washing reagent 12. Next, for example, the magnet is brought close to the side wall of the first extraction container, and the supernatant is discarded so that the nucleic acid-binding solid phase carrier 20 does not spill from the first extraction container. Note that the first cleaning step may be repeated a plurality of times.

Next, the nucleic acid-binding solid phase carrier 20 to which the nucleic acid has been adsorbed is placed in the second nucleic acid washing reagent 13, and the nucleic acid-binding solid phase carrier 20 to which the nucleic acid has been adsorbed is washed (step S3, second washing step). Specifically, the second nucleic acid washing reagent 13 is taken out from the container 32 and introduced into the first extraction container, and the nucleic acid-binding solid phase carrier 20 is placed in the second nucleic acid washing reagent 13 and then stirred. Thereby, the nucleic acid-binding solid phase carrier 20 can be washed with the second nucleic acid washing reagent 13. Next, for example, the magnet is brought close to the side wall of the first extraction container, and the supernatant is discarded so that the nucleic acid-binding solid phase carrier 20 does not spill from the first extraction container. Note that the second cleaning step may be repeated a plurality of times.

  Next, the nucleic acid-binding solid phase carrier 20 to which the nucleic acid has been adsorbed is placed in the third nucleic acid washing reagent 14, and the nucleic acid-binding solid phase carrier 20 to which the nucleic acid has been adsorbed is washed (step S4, third washing step). Specifically, the third nucleic acid washing reagent 14 is taken out from the container 33 and introduced into the first extraction container, and the nucleic acid-binding solid phase carrier 20 is placed in the third nucleic acid washing reagent 14 and then stirred. Thereby, the nucleic acid-binding solid phase carrier 20 can be washed with the third nucleic acid washing reagent 14. Next, for example, the magnet is brought close to the side wall of the first extraction container, and the supernatant is discarded so that the nucleic acid-binding solid phase carrier 20 does not spill from the first extraction container.

  Next, the nucleic acid-binding solid phase carrier 20 on which the nucleic acid has been adsorbed is placed in the nucleic acid elution reagent 15 and the nucleic acid is eluted (step S5, elution step). Specifically, the nucleic acid elution reagent 15 is taken out from the container 34 and introduced into the first extraction container, and the nucleic acid-binding solid phase carrier 20 is placed in the nucleic acid elution reagent 15 and then stirred. Thereby, the nucleic acid can be separated from the nucleic acid-binding solid phase carrier 20 to which the nucleic acid has been adsorbed, and the nucleic acid can be eluted in the nucleic acid elution reagent 15. Next, for example, the supernatant containing the nucleic acid is removed from the second extraction container (the first extraction container and the first extraction container) so that the magnet is brought close to the side wall of the first extraction container and the nucleic acid-binding solid phase carrier 20 does not spill from the first extraction container. Collect in a different container.

  Nucleic acids can be extracted and recovered by the above steps. One of the first cleaning process and the second cleaning process may be omitted.

3. Experimental Example An experimental example is shown below to describe the present invention more specifically. The present invention is not limited by the following experimental examples.

3.1. Experimental method 3.1.1. Nucleic acid extraction method (1) When the target nucleic acid is RNA 62% by mass of guanidine thiocyanate, 1.9% by mass of Triton X-100, 0.58% by mass of Tris (pH 7.2) The nucleic acid-dissolving adsorption reagent was prepared by adding 50% v / v 100% ethanol to the aqueous solution.

  The first nucleic acid washing reagent was prepared by adding 57% v / v 100% ethanol to an aqueous solution containing 61% by mass of guanidine hydrochloride.

  The second nucleic acid washing reagent was prepared by adding 100 v ethanol to 70 v / v% to an aqueous solution containing 0.38 wt% Tris (pH 7.5) and 0.77 wt% NaCl.

  The third nucleic acid washing reagent was prepared by adding 0.05 v / v% of an aqueous solution containing 20% Triton X-100 to an aqueous solution containing 4.9 mM citric acid (pH 4.0).

  Sterile water was used as the nucleic acid elution reagent.

Into the first eppen, 700 μL of the nucleic acid dissolving / adsorbing reagent was introduced, and 140 μL of the sample container containing RNA as the target nucleic acid was further introduced, stirred (vortexed) for 15 seconds, and allowed to settle (spin down). As the target nucleic acid, cultured influenza A virus (I
What was obtained from nfA) was used. Next, 25 μL of a liquid containing magnetic beads (MagExtractor Viral RNA) coated with silica on the surface was added to the first eppen, and the mixture was allowed to settle after 10 stirring. Next, the first eppen was set on a magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (dissolution adsorption step).

  Next, 450 μL of the first nucleic acid washing reagent was added to the first eppen, stirred for 5 seconds, and then allowed to settle. Next, the first eppen was set on the magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (first washing step).

  Next, 450 μL of the second nucleic acid washing reagent was added to the first Eppen, stirred for 5 seconds, and then allowed to settle. Next, the first eppen was set on the magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (second washing step).

  Next, 350 μL of the above third nucleic acid washing reagent was added to the first Eppen, stirred for 5 seconds, and then allowed to settle. Next, the first eppen was set on the magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (third washing step).

  Next, 27.5 μL of the above nucleic acid elution reagent was added to the first eppen, stirred for 5 seconds, and then placed in a tube heater set at 95 ° C. for 2 minutes. Next, after stirring for 5 seconds, it was allowed to settle. Next, when the first eppen was set on a magnetic stand and the magnetic beads were collected, the supernatant was collected in a second eppen (an eppen different from the first eppen) (elution step).

(2) When the nucleic acid is DNA An aqueous solution containing 62% by mass of guanidine thiocyanate, 1.9% by mass of Triton X-100, and 0.58% by mass of Tris (pH 7.2) A dissolved adsorption reagent was used.

  An aqueous solution containing 7.3M guanidine hydrochloride was used as the first nucleic acid washing reagent.

  70 v / v% ethanol obtained by adding 70 mL of ethanol to 30 mL of water was used as the second nucleic acid washing reagent.

  A third nucleic acid washing reagent was prepared by adding 0.05 v / v% of an aqueous Triton X-100 solution to an aqueous solution containing 4.9 mM citric acid (pH 4.0).

  Sterile water was used as the nucleic acid elution reagent.

  750 μL of the above-mentioned dissolved adsorption reagent was introduced into the first eppen, and further 100 μL of the sample container containing DNA as the target nucleic acid was mixed with the dissolved adsorption reagent, stirred for 15 seconds, and then allowed to settle. As the target nucleic acid, one obtained from cultured E. coli was used. Next, 40 μL of a liquid containing magnetic beads coated with silica (MagExtractor Viral RNA) was added to the first eppen, and the mixture was stirred for 10 minutes and then allowed to settle. Next, the first eppen was set on a magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (dissolution adsorption step).

  Next, 900 μL of the first nucleic acid washing reagent was added to the first Eppen, stirred for 5 seconds, and then allowed to settle. Next, the first eppen was set on the magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (first washing step).

  Next, 900 μL of the second nucleic acid washing reagent was added to the first Eppen, stirred for 5 seconds, and then allowed to settle. Next, the first eppen was set on the magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (second washing step).

  Next, 900 μL of the third nucleic acid washing reagent was added to the first Eppen, stirred for 5 seconds, and then allowed to settle. Next, the first eppen was set on the magnetic stand, and when the magnetic beads were collected, the supernatant was discarded (third washing step).

  Next, 100 μL of the above nucleic acid elution reagent was added to the first eppen and stirred for 10 minutes, and then the first eppen was placed in a tube heater set at 95 ° C. and allowed to stand for 2 minutes. Next, after stirring for 5 seconds, it was allowed to settle. Next, when the first eppen was set on a magnetic stand and the magnetic beads were collected, the supernatant was collected in a second eppen (an eppen different from the first eppen) (elution step).

3.1.2. Conditions for real-time PCR (1) When the target nucleic acid is RNA 2 μL of the extract containing RNA extracted and recovered by the above method is added to 8 μL of the PCR solution (master mix), and 10 μL of the reaction solution Was prepared. The composition of the reaction solution is shown below.

<Composition of reaction solution>
Forward primer (20μM) 0.4μL
Reverse primer (20μM) 0.4μL
Fluorescently labeled probe (10 μM) 0.2 μL
Reverse transcriptase / polymerase 0.2 μL
Reaction mixture (Reaction Mix) 5.0 μL
1.8 μL of water
Extract 2.0 μL

  A TaqMan (registered trademark) probe manufactured by Sigma Aldrich was used as a fluorescently labeled probe. In addition, Super Script III Platinum manufactured by Invitrogen was used as reverse transcriptase / polymerase. The reaction mixture is a liquid containing a buffer and dNTP.

  Real-time PCR was performed using One-Step qRT-PCR System manufactured by Invitrogen. In real-time PCR, the reaction solution was heated at 50 ° C. for 30 minutes, then heated at 95 ° C. for 2 minutes, and then a temperature cycle was applied to the reaction solution. The temperature cycle is one cycle consisting of heating at 95 ° C. for 15 seconds and heating at 55 ° C. for 30 seconds, and the reaction solution was given 50 temperature cycles.

(2) When the target nucleic acid is DNA 2 μL of the extract containing the DNA extracted and recovered by the above method was added to 8 μL of the PCR solution (master mix) to prepare 10 μL of a reaction solution. The composition of the reaction solution is shown below.

<Composition of reaction solution>
Forward primer (20μM) 0.4μL
Reverse primer (20μM) 0.4μL
Fluorescently labeled probe (10 μM) 0.2 μL
Polymerase 0.2 μL
Buffer 2.0 μL
dNTP (10 mM) 0.25 μL
Water 4.55μL
Extract 2.0 μL

A TaqMan probe manufactured by Sigma Aldrich was used as the fluorescent labeling probe. As the polymerase, Platinum manufactured by Invitrogen was used. Further, the buffer contains Tris (250 mM) at pH 9.0, KCl (125 mM), and MgCl ₂ (25 mM).

  In real-time PCR, the reaction solution was heated at 95 ° C. for 2 minutes, and then a temperature cycle was applied to the reaction solution. The temperature cycle is one cycle consisting of heating at 95 ° C. for 5 seconds and heating at 70 ° C. for 20 seconds, and the reaction solution was given 50 temperature cycles.

3.2. First Experiment The nucleic acid recovery efficiency was evaluated by real-time PCR on the extract containing the nucleic acid extracted and recovered by the above method (RNA or DNA as the target nucleic acid). A comparison was made between the above-mentioned dissolved adsorption reagent to which 1 mg of bovine serum albumin (BSA) was added (“with BSA”) and the one to which BSA was not added (“without BSA”). In the first experiment, when the RNA was extracted and recovered, each of the first washing step and the second washing step was performed twice.

  FIG. 3 is a graph showing Ct values relative to the presence or absence of BSA when the nucleic acid is RNA. FIG. 4 is a graph showing the Ct value relative to the presence or absence of BSA when the nucleic acid is DNA. The Ct value is the number of temperature cycles when the fluorescence intensity shows a constant value in real-time PCR. The smaller the Ct value, the more nucleic acid can be recovered.

  As shown in FIG. 3 and FIG. 4, in both RNA and DNA, the Ct value was smaller with BSA than without BSA. Therefore, it was found that the nucleic acid dissolving / adsorbing reagent containing BSA can extract nucleic acids more efficiently than the nucleic acid dissolving / adsorbing reagent not containing BSA.

3.3. Second Experiment The nucleic acid recovery efficiency was evaluated by real-time PCR on the extract containing the nucleic acid extracted and recovered by the above method (RNA that is the target nucleic acid). Specifically, the influence of the added amount of BSA was evaluated. More specifically, with respect to 700 μL of the nucleic acid dissolution adsorption reagent, the amount of BSA added is 0.001 mg (0.00143 mg / mL), 0.01 mg (0.0143 mg / mL), 0.1 mg (0.143 mg). / ML), 1 mg (1.43 mg / mL), 10 mg (14.3 mg / mL), and zero (no addition). In the second experiment, the first cleaning step and the second cleaning step were each performed twice.

  FIG. 5 is a graph showing the Ct value versus the amount of BSA added when the nucleic acid is RNA. From FIG. 5, the amount of BSA added is 0.001 mg to 10 mg, preferably 0.01 mg to 10 mg, more preferably 0.1 mg to 10 mg, and even more preferably 0.1 mg to 1 mg. It was found that nucleic acids can be extracted more efficiently.

3.4. Third Experiment In the third experiment, when the nucleic acid (RNA that is the target nucleic acid) is extracted by the above method, the supernatant is not discarded and the nucleic acid extraction kit (QIAHamp viral RNA) manufactured by Qiagen is used. The nucleic acid in the supernatant is collected and PCR is performed, and the number of copies of the nucleic acid contained in the supernatant is evaluated in each step of the dissolution adsorption process, the first washing process, the second washing process, the third washing process, and the elution process. did. A comparison was made between the above-mentioned dissolved adsorption reagent with 1 mg of BSA added and one without BSA added.

  FIG. 6 is a graph showing the copy number in each step when the nucleic acid is RNA. The copy number is the number of nucleic acids amplified by PCR. A Ct value is obtained by performing PCR on the supernatant of each step, and a calibration curve relating to the copy number and Ct value prepared in advance from the CT value. It is obtained using

  FIG. 6 shows the copy number of the nucleic acid contained in the supernatant in each step. The smaller the copy number in the dissolution adsorption step and the first to third washing steps, the higher the copy number in the elution step, the more efficient Nucleic acids can be extracted. The “total” on the horizontal axis in FIG. 6 is the total number of nucleic acid copies contained in the supernatant of each step.

  As shown in FIG. 6, in the dissolution adsorption process, the first washing process, and the second washing process, there was no significant difference in the number of copies with or without BSA, but in the third washing process, no BSA was detected when BSA was present. The copy number was very large compared to that, and in the elution step, the copy number was much larger with BSA than without BSA. From this, it was found that without BSA, many nucleic acids were detached from the magnetic beads in the third washing step and the copy number in the elution step was reduced. Therefore, it was found that when BSA was added to the nucleic acid dissolving / adsorbing reagent, it was possible to suppress the nucleic acid from desorbing from the magnetic beads in the third washing step, and to efficiently extract the nucleic acid in the elution step.

  Here, the first nucleic acid washing reagent and the second nucleic acid washing reagent contain a chaotropic agent, but the third nucleic acid washing reagent does not contain a chaotropic agent and is acidic. Chaotropic agents inhibit PCR, but acidic liquids are difficult to inhibit PCR. Therefore, if BSA is added to the dissolved adsorption reagent, even if an acidic third nucleic acid washing reagent that does not contain a chaotropic agent is used, it is possible to prevent nucleic acids from being detached from the magnetic beads in the third washing step, Furthermore, since the third nucleic acid washing reagent is difficult to inhibit PCR, the fluorescence intensity in real-time PCR can be increased and the sensitivity can be improved.

3.5. Discussion As described above, it was found that when BSA was added to the nucleic acid dissolution / adsorption reagent, nucleic acid could be prevented from being detached from the magnetic beads in the third washing step, and the nucleic acid could be extracted efficiently in the elution step. Consider the reason. In addition, the experiment which supports the following consideration was not conducted.

  Chaotropic agents adsorb nucleic acids on the surface of magnetic beads by hydrophobic interaction. Ethanol also has a hydrophobic interaction. The third nucleic acid washing reagent contains neither a chaotropic agent nor ethanol. Therefore, when BSA is not added to the nucleic acid dissolution and adsorption reagent, the nucleic acid is detached from the magnetic beads in the third washing step.

Here, FIG. 7 is a graph for explaining the relationship between the pH of silica (SiO ₂ ), BSA, and nucleic acid (RNA, DNA) and the charge. FIG. 8 is a cross-sectional view schematically showing a state in which bovine serum albumin (BSA) 2 is adsorbed to a nucleic acid-binding solid phase carrier (magnetic bead) 20 having metal particles 20a and a silicon oxide (silica) layer 20b. is there.

  As shown in FIG. 7, silica is a substance whose charge changes when pH is changed, and the isoelectric point (pH at which the charge becomes zero) is around pH = 2. On the other hand, nucleic acids are negatively charged for any pH.

  Since the surface of the magnetic bead is silica, when the pH is greater than 2, the surface of the magnetic bead is negatively charged. Since the nucleic acid is also negatively charged, the nucleic acid is detached from the surface of the magnetic bead.

  As shown in FIG. 8, BSA2 has a property of causing a physical hydrophobic interaction (physical adsorption) on the surface of the magnetic bead 20. Therefore, for example, even if the surface of the magnetic bead 20 is negatively charged and the BSA2 is also negatively charged, the BSA2 can be adsorbed on the surface of the magnetic bead.

  As shown in FIG. 7, BSA is a substance whose charge changes when pH is changed, and its isoelectric point is around pH = 4.7. Therefore, BSA charges positively by setting the pH to less than 4.7. Thereby, the negatively charged nucleic acid can be adsorbed to the magnetic beads adsorbed with BSA. That is, the negatively charged nucleic acid is adsorbed on the surface of the magnetic beads via the positively charged BSA. Therefore, when BSA is added to the dissolved adsorption reagent, nucleic acid can be prevented from being detached from the magnetic beads in an acidic environment in the third washing step. In the elution step, BSA is negatively charged in a neutral environment, and the nucleic acid can be detached from the magnetic beads.

  Therefore, instead of BSA, a substance that physically adsorbs on magnetic beads whose surface is silica, charges positively in an acidic environment, and charges negatively in a neutral environment is added to the nucleic acid-dissolving adsorption reagent. Even so, it is inferred that the same effect as BSA can be obtained. Such substances include skim milk, gelatin, casein, globulin, and albumin that is not bovine serum.

  The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

2 ... Bovine serum albumin, 10 ... Nucleic acid extraction reagent set, 11 ... Nucleic acid dissolution and adsorption reagent, 12 ... First nucleic acid washing reagent, 13 ... Second nucleic acid washing reagent, 14 ... Third nucleic acid washing reagent, 15 ... Nucleic acid elution reagent, DESCRIPTION OF SYMBOLS 20 ... Nucleic acid binding solid-phase carrier, 20a ... Metal particle, 20b ... Silicon oxide layer, 22 ... Liquid, 30, 31, 32, 33, 34, 35 ... Container, 40 ... Case, 100 ... Nucleic acid extraction kit

Claims (8)

A chaotropic agent and an additive,
The additive includes at least one of albumin, skim milk, gelatin, casein, and globulin,
A nucleic acid dissolution and adsorption reagent that dissolves nucleic acid and adsorbs the dissolved nucleic acid to a nucleic acid-binding solid phase carrier whose surface is silicon oxide. In claim 1,
The nucleic acid-dissolving adsorption reagent, wherein the additive is bovine serum albumin. In claim 1 or 2,
The nucleic acid dissolving / adsorbing reagent, wherein the concentration of the additive contained in the nucleic acid dissolving / adsorbing reagent is 0.001 mg / mL to 15 mg / mL. In any one of Claims 1 thru | or 3,
The nucleic acid dissolving / adsorbing reagent, wherein the concentration of the additive contained in the nucleic acid dissolving / adsorbing reagent is 0.1 mg / mL to 1.5 mg / mL. A nucleic acid dissolving / adsorbing reagent according to any one of claims 1 to 4,
An acidic nucleic acid washing reagent for washing the nucleic acid-binding solid phase carrier adsorbed with nucleic acid;
A nucleic acid elution reagent for separating and eluting nucleic acid from the washed nucleic acid-binding solid phase carrier;
A nucleic acid extraction reagent set comprising: In claim 5,
The nucleic acid extraction reagent set, wherein the pH of the nucleic acid washing reagent is less than 4.7. A first container in which the nucleic acid dissolving / adsorbing reagent according to any one of claims 1 to 4 is accommodated;
A second container containing an acidic nucleic acid washing reagent for washing the nucleic acid binding solid phase carrier to which the nucleic acid has been adsorbed;
A third container containing a nucleic acid elution reagent for separating and eluting nucleic acid from the washed nucleic acid binding solid phase carrier;
A nucleic acid extraction kit comprising: A step of dissolving a nucleic acid in a nucleic acid dissolution and adsorption reagent containing a chaotropic agent and an additive, and adsorbing the dissolved nucleic acid to a nucleic acid-binding solid phase carrier whose surface is silicon oxide;
Placing the nucleic acid binding solid phase carrier adsorbed with nucleic acid in an acidic nucleic acid washing reagent, and washing the nucleic acid binding solid phase carrier adsorbed with nucleic acid;
After the washing step, placing the nucleic acid-binding solid phase carrier adsorbed with nucleic acid in a nucleic acid elution reagent, and eluting the nucleic acid;
Including
The nucleic acid extraction method, wherein the additive comprises at least one of albumin, skim milk, gelatin, casein, and globulin.

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