JP2010158190A - Method and apparatus for collecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To collect nucleic acids stably or with constant excellent efficiency by improving the deterioration of nucleic acid-adsorbing ability in a nucleic acid-bondable carrier. <P>SOLUTION: The method for collecting nucleic acids by bringing a solution containing the nucleic acids into contact with the nucleic acid-bondable carrier to allow the nucleic acid-bondable carrier to adsorb the nucleic acids includes a step of bringing the nucleic acid-bondable carrier into contact with a liquid or for exposing the carrier to high-temperature and high-pressure conditions before the contact of the nucleic acid-bondable carrier with the solution containing the nucleic acids. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a nucleic acid recovery method and a nucleic acid recovery apparatus for eluting nucleic acid from a solid phase bound with nucleic acid and recovering the nucleic acid in a solution.

  Analysis of nucleic acids, which are substances that carry genetic information, is carried out in various fields such as academic research and medical treatment. Nucleic acid amplification techniques such as PCR (Polymerase Chain Reaction) are often used as nucleic acid analysis methods. PCR is a method for amplifying nucleic acid in a base sequence-specific manner, and enables detection and quantification of a very small amount of a target gene. Nucleic acids extracted from biological samples are used for nucleic acid analysis such as PCR. In this nucleic acid extraction, contaminants that contain inhibitors for analytical reactions, such as nucleic acids derived from the external environment that give false positive results in the analysis results. It is necessary to eliminate the influence of.

  As a method for extracting nucleic acid from a biological sample or the like, there is a conventional method in which an operation such as ethanol precipitation is performed using a poisonous or deleterious substance such as phenol or chloroform, but in recent years, as described in Non-Patent Document 1. A method that utilizes the property of nucleic acid binding to a silica-containing material or the like in the presence of a chaotropic material is mainly employed. Furthermore, various improvements have been made to nucleic acid-binding carriers such as silica-containing substances for the purpose of simplification or automation. For example, in Patent Document 1, a cylindrical member holding a nucleic acid-binding carrier at the bottom, Patent Document 2 is a cartridge having glass powder, Patent Document 3 is a chip that holds a nucleic acid-binding carrier in a predetermined state, etc. Has been made.

J. Clin. Microbiol., 28 (3), 495-503 (1990) Patent No. 2936111 Patent No. 3045922 Japanese Patent No. 3619514

  The methods described in Patent Documents 1 to 3 have various improvements in terms of simplification or automation, but considerations regarding stabilization of performance have not always been sufficient. That is, the nucleic acid binding carrier that adsorbs nucleic acids has a problem in that the nucleic acid adsorption ability varies and the nucleic acid adsorption ability decreases with time. For example, when the nucleic acid binding carrier is left in a normal indoor environment, there is a problem that the nucleic acid binding performance is lowered.

  Therefore, in view of the above-described circumstances, the present invention provides a nucleic acid recovery method and a nucleic acid recovery method that can recover nucleic acid stably or always with excellent efficiency by improving degradation of nucleic acid adsorption ability in a nucleic acid binding carrier. An object is to provide an apparatus.

  As a result of intensive studies by the present inventor in order to achieve the above-described object, it has been revealed for the first time that the decrease in the nucleic acid-adsorbing ability of the nucleic acid-binding carrier is caused by the change in the surface properties of the nucleic acid-binding carrier. In addition, it was clarified that deterioration of nucleic acid adsorption ability can be improved by improving the surface properties of the nucleic acid binding carrier before adsorbing the nucleic acid to the nucleic acid binding carrier, and the present invention has been completed.

That is, the present invention includes the following.
The method for recovering nucleic acid according to the present invention is a method for recovering nucleic acid by bringing a nucleic acid-binding carrier into contact with a solution containing the nucleic acid and adsorbing the nucleic acid to the nucleic acid-binding carrier. Prior to the contact between the nucleic acid-binding carrier and the nucleic acid-binding carrier, a step of bringing a liquid into contact with the nucleic acid-binding carrier or exposing to a high temperature and high pressure condition is provided.

  In the nucleic acid recovery method according to the present invention, examples of the nucleic acid binding carrier include substances containing silicon oxide. Examples of the liquid include alkaline solutions and organic solvents. Further, as the liquid, two or more kinds of liquids having different compositions may be used, and the two or more kinds of liquids may be sequentially brought into contact with the nucleic acid binding carrier. Examples of the nucleic acid include ribonucleotides.

  The nucleic acid recovery method according to the present invention comprises a step of bringing a liquid or a high-temperature high-pressure gas into contact with the nucleic acid-binding carrier, and then bringing the nucleic acid-containing liquid into contact with the nucleic acid-binding carrier; It may further include a step of bringing the adsorbed nucleic acid-binding carrier into contact with the eluent and eluting the nucleic acid in the eluent.

  On the other hand, the nucleic acid recovery apparatus according to the present invention includes an extraction column provided with a nucleic acid binding carrier, a dispensing mechanism for feeding a solution into the extraction column, and a solution fed into the extraction column discharged through the inside of the nucleic acid binding carrier. A nucleic acid extraction apparatus including a pressurizing mechanism for contacting a liquid with a nucleic acid-binding carrier or exposing to a high-temperature and high-pressure condition prior to the contact between the nucleic acid-containing solution and the nucleic acid-binding carrier. .

  Moreover, the structure provided with the container holding the said liquid may be sufficient as the nucleic acid collection | recovery apparatus which concerns on this invention. In this case, in the nucleic acid recovery apparatus according to the present invention, the dispensing mechanism puts the liquid in the container into the extraction column, and the pressurizing mechanism sends the liquid put into the extraction column into the nucleic acid-binding carrier. The liquid can be brought into contact with the nucleic acid-binding carrier by discharging it through.

  ADVANTAGE OF THE INVENTION According to this invention, the fall of the nucleic acid adsorption ability in a nucleic acid binding support | carrier can be improved, and the nucleic acid adsorption | suction efficiency using a nucleic acid binding support | carrier can be stabilized. Therefore, according to the nucleic acid recovery method and the nucleic acid recovery apparatus according to the present invention, the nucleic acid binding ability of the nucleic acid binding carrier is stably exerted without being affected by the storage state or storage period of the nucleic acid binding carrier, and the nucleic acid is recovered. It can be recovered.

Hereinafter, the present invention will be described in detail with reference to the drawings.
In the nucleic acid recovery method according to the present invention, first, prior to the step of adsorbing nucleic acid to the nucleic acid binding carrier, a liquid is brought into contact with the nucleic acid binding carrier or exposed to high temperature and high pressure conditions. In other words, the nucleic acid recovery method according to the present invention includes a step of bringing a liquid into contact with a nucleic acid-binding carrier or exposing to a high-temperature and high-pressure condition, and a step of adsorbing a nucleic acid to the nucleic acid-binding carrier after the step. It is the method of including.

  In the following description, “to make a liquid contact with a nucleic acid-binding carrier or to be exposed to high-temperature and high-pressure conditions” means “to wash a nucleic acid-binding carrier” or “to perform a washing step of a nucleic acid-binding carrier”. Called.

  Here, any nucleic acid-binding carrier can be used as long as it contains silica such as glass or diatomaceous earth. Alternatively, examples of the nucleic acid binding carrier include organic substances such as acetyl cellulose and conventionally known substances such as magnetic beads. These substances can be processed into fine particles or porous membranes to form a solid phase. In particular, the nucleic acid-binding carrier is preferably a silica-containing substance because the nucleic acid adsorption ability can be greatly recovered by the washing step described above.

  The liquid used for washing (hereinafter sometimes referred to as a washing liquid) is not particularly limited as long as it has a nucleic acid binding ability recovery action on the nucleic acid binding carrier. Preferable examples of the cleaning liquid include pure water, ionic aqueous solutions such as hydrochloric acid aqueous solution and sodium hydroxide aqueous solution, organic solvents such as xylene, and mixed solutions thereof. These washing solutions are preferred because even if they remain on the nucleic acid-binding carrier, they do not adversely affect the next step described later.

  When the nucleic acid-binding carrier is washed using the above-described washing liquid, it is not particularly limited, but the washing liquid may be brought into contact with the nucleic acid-binding carrier. By bringing the washing solution into contact with the nucleic acid binding carrier, the surface property of the nucleic acid binding carrier is changed and the nucleic acid adsorption ability is recovered. More specifically, for example, when a nucleic acid-binding carrier is stored, a gas component contained in the storage environment may be adsorbed on the surface, and the nucleic acid adsorption ability may deteriorate due to this. By bringing the washing solution and the nucleic acid binding carrier into contact with each other, for example, gas components adsorbed on the surface of the nucleic acid binding carrier can be removed, and the nucleic acid adsorption ability can be recovered.

  Further, when the nucleic acid-binding carrier is brought into contact with the washing liquid, the washing liquid may be passed through a column containing the nucleic acid-binding carrier. For example, when using a porous membrane-like nucleic acid-binding carrier, an instrument in which the porous membrane-like nucleic acid-binding carrier is fixed inside a hollow pipe is used. The porous membrane-like nucleic acid binding carrier can also be fixed so as to be sandwiched between a pair of solid phase holding members inside the pipe. At this time, the pair of solid phase holding members can be formed of, for example, a porous polypropylene particle sintered body. When this instrument is used, one end of the pipe is an open end, the other end is connected to a pump device, and the pump device is driven to suck and discharge the solution from the one end. Thereby, the washing solution can be brought into contact with the porous membrane-like nucleic acid binding carrier. It should be noted that the solution sucked into the instrument can be discharged using centrifugal force.

On the other hand, the washing step is not limited to the above-described method in which the washing solution is brought into contact with the nucleic acid-binding carrier, and may be a process of exposing the nucleic acid-binding carrier to high temperature and high pressure conditions using an autoclave or the like. By exposing the nucleic acid-binding carrier to high-temperature and high-pressure conditions, the surface property of the nucleic acid-binding carrier is changed and the nucleic acid adsorption ability is recovered. The high temperature and high pressure conditions are not particularly limited as long as the nucleic acid adsorption ability of the nucleic acid binding carrier can be recovered. More specifically, the temperature condition is preferably 45 to 135 ° C, more preferably 105 to 121 ° C, and most preferably 121 ° C. The pressure condition is preferably 1 × 10 ^{5 to} 2.5 × 10 ⁵ Pa, and most preferably 2 × 10 ⁵ Pa.

  The nucleic acid-binding carrier can be adsorbed on the nucleic acid-binding carrier by bringing the sample containing the nucleic acid into contact with the nucleic acid-binding carrier after washing as described above. Here, examples of the nucleic acid-containing sample include whole blood, plasma, serum, urine, stool, saliva, sputum, semen and other body fluids, animal and plant tissues and cells, microorganisms, bacteria, viruses and the like, and suspensions thereof. Or dissolved product can be used. When adsorbing a nucleic acid on a nucleic acid binding carrier, it is preferable to use a so-called binding reagent that promotes adsorption of the nucleic acid. The binding reagent is not particularly limited as long as it can liberate the nucleic acid in the nucleic acid-containing sample and promote the binding between the nucleic acid and the nucleic acid binding carrier. For example, various chaotropic salts can be used as the binding reagent. As the chaotropic salt, guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate and the like can be used. In addition, a surfactant or a proteolytic enzyme can be used to promote the liberation of nucleic acid. As the surfactant, a nonionic surfactant is preferable, and polyoxyethylene, octylphenyl ether, polyoxyethylene, sorbitan monolaurate and the like can be used. In addition, reducing agents such as 2-mercaptoethanol and dithiothreitol and metal chelating agents such as ethylenediaminetetraacetate can be used to inactivate nucleolytic enzymes that are inherent in nucleic acid-containing samples or derived from the external environment. . Moreover, in order to accelerate | stimulate liberation of a nucleic acid, processes, such as a heating, stirring, a homogenization, and an ultrasonic wave, can be used together. In addition, an organic solvent can be used to promote the binding between the nucleic acid and the nucleic acid binding carrier. The organic solvent is preferably a water-soluble organic solvent, and methanol, ethanol, isopropanol, butanol, acetone, diethylene glycol dimethyl ether, ethyl lactate, and the like can be used.

  As an example of an extraction device including a nucleic acid binding carrier, a schematic diagram of an extraction column is shown in FIG. As shown in FIG. 1, the extraction column includes an opening 21 for introducing a solution, a connection 22 for connecting a pressurizing mechanism, and an opening 23 for discharging the solution. It is fixed by the holding member 25. The nucleic acid-binding carrier 24 shown in FIG. 1 is a solid phase containing, for example, glass, silica, diatomaceous earth, or acetylcellulose, and has a plurality of continuous liquid passage holes and can be passed therethrough. There can be used a fine and tight filter-like structure so that sufficient contact efficiency with the solution can be obtained when the liquid is passed. As the holding member 25, a member that can maintain the shape and the installation position of the nucleic acid binding carrier 24 and that has a plurality of continuous liquid passage holes and can be used can be used. A ligature etc. can be used.

  FIG. 2 shows a pressurizing mechanism for pressurizing the inside of the extraction column into which the solution is charged and discharging the solution through the inside of the nucleic acid binding carrier 24. The pressurizing mechanism includes a syringe 31 and a plunger 32 for pressurizing the inside of the extraction column, a plunger driving motor 33 for driving the plunger 32 up and down, and a syringe for driving the syringe 31 and the plunger driving motor 33 up and down. The drive motor 34, a seal member 36 disposed between the syringe 31 and the extraction column 35, an extraction column holding stand 37, a waste liquid receiver 38, and a waste liquid receiver holding stand 39 are configured. In this pressurizing mechanism, first, the plunger 32 and the syringe 31 are moved upward, and the extraction column 35 and the waste liquid receiver 38 are installed in the extraction column holding stand 37 and the waste liquid receiver holding stand 39, respectively. Next, in this pressurizing mechanism, the above-described solution is introduced into the extraction column 35, the syringe 31 is moved downward, and the syringe 31 and the extraction column 35 are brought into close contact with each other via the seal member 36. Next, in this pressurizing mechanism, the plunger 32 is moved downward, the inside of the extraction column 35 is pressurized, and the solution in the extraction column 35 is discharged to the waste liquid receiver 38 through the inside of the nucleic acid binding carrier.

  Note that the extraction column shown in FIG. 1 and the pressurization mechanism shown in FIG. 2 can be used in the same manner when the cleaning solution described above is brought into contact with the nucleic acid binding carrier. In addition, after the nucleic acid is adsorbed on the nucleic acid binding carrier, for example, the first washing step, the second washing step, and the elution step are performed as follows.

  As the first washing solution, the binding between the nucleic acid and the nucleic acid binding carrier can be maintained, and non-specific binding substances such as proteins can be removed. Chaotropic salts, surfactants, which can be used in the binding solution, A solution containing a reducing agent, a metal chelating agent, an organic solvent and the like can be used. As the second washing liquid, the binding between the nucleic acid and the nucleic acid binding carrier is maintained, the non-specific binding substance that could not be removed by the first washing liquid and the residual components of the first washing liquid are removed, and the next step A solution containing a volatile organic solvent such as ethanol or acetone can be used. As a method for removing the residual liquid of the second washing liquid, a method of heating and drying the nucleic acid-binding carrier portion of the extraction column can be used.

  The eluate is one that elutes nucleic acid from a nucleic acid binding carrier and does not contain components that inhibit the subsequent analysis reaction, and uses pure water or low salt concentration buffer that does not contain nucleolytic enzymes. it can. In addition, the eluate can be heated in order to promote the elution of nucleic acid. The subsequent analysis reaction for the extracted nucleic acid includes PCR (Polymerase Chain Reaction), RT-PCR (Reverse Transcription-Polymerase Chain Reaction), LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification ), NASBA (; Nucleic Acid Sequence-Based Amplification), TMA (; Transcription-Mediated Amplification), LAMP (; Loop-mediated isothermal amplification), ICAN (; Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) In addition, nucleic acid analysis such as reverse transcription reaction, nucleic acid extension reaction, enzyme reaction such as restriction enzyme, or electrophoresis can be applied.

  As described above, according to the present invention, even if the nucleic acid adsorption ability of the nucleic acid binding carrier is deteriorated, the nucleic acid adsorption ability can be recovered. Therefore, by performing the above-described washing step prior to the step of adsorbing the nucleic acid to the nucleic acid binding carrier, the nucleic acid can be recovered stably or always with excellent efficiency.

  The interval between the washing step and the nucleic acid adsorption step described above is not particularly limited, but is preferably within 3 days after the washing step, more preferably within 1 day, and most preferably within 4 hours. When the nucleic acid adsorption step is performed after one day after the completion of the washing step, the nucleic acid binding carrier may be deteriorated again. When the nucleic acid-binding carrier can be sealed and stored in an environment where the adsorbed gas is removed after completion of the washing step, the nucleic acid-binding ability of the nucleic acid-binding carrier can be maintained at a high level. Then, the nucleic acid adsorption step may be performed.

  Hereinafter, the nucleic acid recovery method and the nucleic acid recovery apparatus according to the present invention will be described using examples, but the technical scope of the present invention is not limited to these examples.

First, a sample, a reagent, a nucleic acid extraction instrument, a nucleic acid extraction method, a nucleic acid quantification method, and a nucleic acid analysis method used in this example will be described in order.
1. Sample Pseudo sample (100ng / 200μl) with total RNA purified from mouse liver added to serum

2. Reagents
Select one from washing solution ultrapure water, sodium chloride, potassium chloride, potassium acetate, hydrochloric acid, aqueous sodium hydroxide, aqueous potassium hydroxide, Tris (pH 8.0) buffer and MES (pH 4.0) buffer. used.

Binding reagent
6M guanidine hydrochloride
100 mM MES
25mM EDTA ・ 2Na
20% (v / v) polyoxyethylene (10) octylphenyl ether

First cleaning solution
2.5M guanidine thiocyanate
100 mM MES
25mM EDTA ・ 2Na
1% (v / v) polyoxyethylene (10) octylphenyl ether

Second cleaning liquid
10 mM Tris-HCl (pH 8.0)
80% (v / v) EtOH

Eluent
10 mM Tris-HCl (pH 8.0)

3. Nucleic acid extraction instrument In the extraction column shown in FIG. 1 described above, the internal volume of the extraction column is 2 ml, the material of the extraction column is polypropylene, the nucleic acid-containing nucleic acid binding carrier is a glass fiber filter paper having an average particle holding diameter of 0.7 μm, holding member Was used as a polypropylene particle sintered body having an average particle holding diameter of about 100 μm.

4. Nucleic acid extraction method The nucleic acid extraction method using the extraction column shown in FIG. 1 and the pressurization mechanism shown in FIG. 2 is as follows.
(1) Install an extraction column and waste receiver in the pressure mechanism.
(2) Put 1 mL of washing solution into the extraction column.
(3) Start the pressure unit.
(4) The syringe is driven downward and comes into close contact with the extraction column via the sealing material.
(5) The plunger is driven downward, and the syringe initial volume 5 ml air phase is compressed to 1 ml.

(6) Discharge the cleaning solution from the bottom of the extraction column.
(7) After a certain period of time, the syringe is driven upward and the connection with the extraction column is released.
(8) Repeat steps (2) to (7). (9) Load the 200 μl sample and 800 μl binding solution into the extraction column.
(10) Start the pressure mechanism.

(11) The syringe is driven downward and is brought into close contact with the extraction column via the sealing material.
(12) The plunger is driven downward, and the syringe initial volume 5 ml air phase is compressed to 1 ml.
(13) The mixed liquid level is detected, the syringe is driven upward, and the connection with the extraction column is released.
(14) The plunger is driven upward.
(15) Add 1200 μl of the first washing solution to the extraction column.

(16) The syringe is driven downward and is brought into close contact with the extraction column via the sealing material.
(17) The plunger is driven downward, and the syringe initial volume 5 ml air phase is compressed to 1 ml.
(18) The cleaning liquid level is detected, the syringe is driven upward, and the connection with the extraction column is released.
(19) The plunger is driven upward.
(20) Add 1200 μl of the second washing solution to the extraction column.

(21) The plunger is driven downward, and the syringe initial volume 5 ml air phase is compressed to 1 ml.
(22) The cleaning liquid level is detected, the syringe is driven upward, and the connection with the extraction column is released.
(23) The plunger is driven upward.
(24) Add 1200 μl of the second washing solution to the extraction column again.
(25) The plunger is driven downward, and the syringe initial volume 5 ml air phase is compressed to 1 ml.

(26) The cleaning liquid level is detected, the syringe is driven upward, and the connection with the extraction column is released.
(27) The plunger is driven upward.
(28) Remove and discard the waste liquid receiver, and install the purified liquid receiver.
(29) Add 110 μl eluate to the extraction column.
(30) The plunger is driven downward, and the syringe initial volume 2 ml air phase is compressed to 1 ml.
(31) The cleaning liquid level is detected, the syringe is driven upward, and the connection with the extraction column is released.

  About 100 μl of eluate was obtained in the purified liquid receptor by the above protocols (1) to (31).

5. Nucleic acid quantification method The nucleic acid after extraction was quantified using RiboGreen, a fluorescent dye, according to the attached procedure manual. Here, the extraction rate was calculated from the recovered RNA amount with the total RNA amount at the time of addition as 100%.

6. Results are shown in FIG. From this result, it has been clarified that the extraction rate of the extraction column left in the environment is remarkably lowered and the extraction efficiency is improved by adding a washing step. Further, from the results shown in FIG. 3, it is clear that there is a difference in the effect of improving the nucleic acid adsorption ability depending on the type of cleaning liquid used in the cleaning step, and that cleaning is particularly effective with a solution containing hydroxide ions. It became.

[Example 1]
In this example, glass fiber filter paper was used as the nucleic acid-binding carrier, and the deterioration over time of the nucleic acid-binding carrier was verified. Specifically, after leaving the nucleic acid-binding carrier to stand for 1 month, the presence or absence of autoclaving conditions at 121 ° C. for 60 minutes, and the number of days (2 days, 5 days and 8 days) after the autoclave treatment, the nucleic acid The change in adsorption capacity was verified.

  The results are shown in FIG. From the results of FIG. 4, it was revealed that the nucleic acid binding carrier deteriorates in its nucleic acid adsorption ability over time and can be recovered by exposure to high temperature and high pressure conditions.

[Example 2]
In this example, an experiment for recovering nucleic acid contained in the sample was performed in the same manner as in Example 1 except that the cleaning liquid was an organic solvent. In this example, the organic solvents include tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), γ-butyl, dimethylformamide (DMF), methidathion (DMTP), tetramethylurea (TMU), dimethylacetamide (DMAc), and dimethyl oleate. Tylene urea (DMEU) was used.

  The results are shown in FIG. From the results shown in FIG. 5, it was clarified that the nucleic acid-adsorbing ability of the nucleic acid-binding carrier can be recovered by bringing the organic solvent into contact with the nucleic acid-binding carrier having degraded nucleic acid-adsorbing ability.

Example 3
In this example, a nucleic acid contained in the sample was collected in the same manner as in Example 1 except that the washing solution was a mixed solution containing an organic solvent and an ionic compound. In this example, as the cleaning liquid, a mixed liquid of 0.1 mol / L sodium chloride and DMEU and a mixed liquid of 0.1 mol / L sodium hydroxide and DMEU were used.

  The results are shown in FIG. From the results shown in FIG. 6, it was revealed that the ability to adsorb nucleic acid can be recovered even when a mixed solution containing an organic solvent and an ionic compound is used as a cleaning solution. In particular, when a mixed solution containing hydroxide ions was used, it was revealed that the degree of recovery of the nucleic acid adsorption ability was more excellent.

Example 4
In this example, the substances adsorbed on the nucleic acid binding carrier were comparatively examined before and after washing. Specifically, first, the extraction column used in Example 1 was left in the laboratory for 1 day in an opened state. Thereafter, procedures (1) to (8) of Example 1 were performed using a predetermined cleaning solution. Then, the extraction column was disassembled, the nucleic acid binding carrier was taken out and analyzed by GC-MS. In this example, a 0.1 mol / L sodium hydroxide solution, a DMEU solution, or a mixed solution of 0.1 mol / L sodium hydroxide and DMEU was used as the cleaning liquid.

  The results are shown in FIG. From the results shown in FIG. 7, it was revealed that the substance adsorbed on the nucleic acid binding carrier was removed by washing. Also, from this result, it was revealed that the recovery of the nucleic acid binding ability of the nucleic acid binding carrier is due to the removal of the substance adsorbed on the nucleic acid binding carrier.

Example 5
According to the above Examples 1 to 4, the nucleic acid adsorption ability of the carrier can be recovered by washing the carrier before adsorbing the nucleic acid to the nucleic acid binding carrier. It became clear that it improved. In this example, it was verified that a nucleic acid analysis method such as a nucleic acid amplification method was feasible even when the concentration of nucleic acid contained in a sample was lowered.

1.Sample
A false positive specimen (10 ³ copies / 200 μl) in which purified DNA from HBV positive serum was added to negative serum was prepared. From this sample, DNA extraction was performed using the various reagents, pressurization mechanism and operation protocol detailed in Example 1.

2.Nucleic acid analysis method
PCR
PCR using HBV-derived DNA as a template DNA was carried out under the following conditions based on the report of (J. Clin. Microbiol., 29 (9), 1804-1811 (1991)). As a template, 10 μl of the eluate prepared in the above 1. sample was used.
Primer 1: 5 'GCG GAT CCG AGT TAC TCT CGT TTT TGC 3' (SEQ ID NO: 1)
Primer 2: 5 'GCA AGC TTT CTA ACA ACA GTA GTT TCC GG 3' (SEQ ID NO: 2)

  The temperature cycle conditions in PCR were first maintained at 94 ° C. for 5 min, then 30 cycles of 94 ° C. for 1 min, 55 ° C. for 1 min and 72 ° C. for 1 min were performed, followed by 72 ° C. for 10 min.

LAMP
Based on the report of (Nucleic Acid Research, 28 (12), e63 (2000)), LAMP using HBV-derived DNA as template DNA was performed under the following conditions. As a template, 10 μl of the eluate prepared in the above 1. sample was used.
Primer FIP: 5 'CCT GCT GCT ATG CCT CAT CTT CTT TGA CAA ACG GGC AAC ATA CCT T 3' (SEQ ID NO: 3)
Primer BIP: 5 'GAT AAA ACG CCG CAG ACA CAT CCT TCC AAC CTC TTG TCC TCC AA 3' (SEQ ID NO: 4)
Primer F3: 5 'GGT GGT TGA TGT TCC TGG A 3' (SEQ ID NO: 5)
Primer B3: 5 'CAA AAT TCG CAG TCC CCA AC 3' (SEQ ID NO: 6)

  The temperature conditions in LAMP were first maintained at 95 ° C. for 5 minutes, then maintained on ice for 3 minutes, added with Bst DNA Polymerase, maintained at 65 ° C. for 1 hour, and then maintained at 80 ° C. for 10 minutes.

Confirmation of amplification products
Electrophoresis using 0.8% agarose gel was performed, stained with a fluorescent dye that specifically binds to double-stranded DNA, and confirmed under ultraviolet irradiation.

Results From the results of electrophoresis, it was revealed that good amplification products were obtained for both the PCR and LAMP described above. From this result, it was proved that normal nucleic acid extraction was performed even from a sample containing a low concentration of nucleic acid, and that it was suitable for the subsequent analysis reaction.

It is a schematic diagram of an extraction column shown as an example of an extraction device provided with a nucleic acid binding carrier. It is a schematic diagram which shows a pressurization mechanism provided with an extraction column. It is a characteristic view which shows the result of having examined the recovery | restoration degree of the nucleic acid adsorption ability by the kind of washing | cleaning liquid. It is a characteristic view which shows the result of having examined the time-dependent deterioration of the nucleic acid adsorption ability in a nucleic acid binding support | carrier. It is a characteristic view which shows the result of having examined the recovery | restoration degree of the nucleic acid adsorption ability by the kind of organic solvent used as a washing | cleaning liquid. It is a characteristic view which shows the result of having examined the recovery | restoration degree of the nucleic acid adsorption ability by the kind of liquid mixture used as a washing | cleaning liquid. It is a characteristic view which shows the result of having compared the substance adsorbed | sucked to the nucleic acid binding support | carrier before and after washing | cleaning.

21 .. Solution loading opening
22. ・ Pressure mechanism connection
23 .. Solution discharge opening
24 ・ ・ Nucleic acid binding solid phase
25 ・ ・ Holding member
31.Syringe
32 ・ Plunger
33 .. Plunger drive motor
34 ・ ・ Syringe drive motor
35 ・ ・ Extraction column
36 ・ ・ Seal material
37 .. Extraction column holding stand
38 ・ ・ Waste liquid receptacle
39 ・ ・ Standing fluid holding stand

Claims (14)

A method of recovering a nucleic acid by bringing a nucleic acid-binding carrier into contact with a solution containing the nucleic acid and adsorbing the nucleic acid to the nucleic acid-binding carrier,
A method for recovering nucleic acid, comprising a step of bringing a liquid into contact with a nucleic acid-binding carrier or exposing to a high-temperature and high-pressure condition prior to contacting the solution containing the nucleic acid with the nucleic acid-binding carrier.   2. The nucleic acid recovery method according to claim 1, wherein the nucleic acid-binding carrier is a substance containing silicon oxide.   The nucleic acid recovery method according to claim 1, wherein the liquid is an alkaline solution.   The nucleic acid recovery method according to claim 1, wherein the liquid contains an organic solvent.   2. The method for recovering nucleic acid according to claim 1, wherein the liquid is a liquid having two or more different compositions, and the two or more liquids are sequentially brought into contact with the nucleic acid binding carrier.   The nucleic acid recovery method according to claim 1, wherein the nucleic acid is a ribonucleotide. A step of bringing a liquid into contact with the nucleic acid-binding carrier or contacting the nucleic acid-binding carrier with a liquid containing a nucleic acid after exposure to a high-temperature and high-pressure condition;
The method for recovering nucleic acid according to claim 1, further comprising a step of bringing the nucleic acid-binding carrier adsorbing the nucleic acid into contact with an eluent and eluting the nucleic acid in the eluent. An extraction column comprising a nucleic acid binding carrier;
A dispensing mechanism for loading the solution into the extraction column;
A nucleic acid extraction device comprising a pressurizing mechanism for discharging the solution charged in the extraction column through the inside of the nucleic acid binding carrier,
A nucleic acid extraction apparatus, wherein a liquid is brought into contact with a nucleic acid-binding carrier or exposed to high-temperature and high-pressure conditions prior to contact between a solution containing nucleic acid and the nucleic acid-binding carrier.   A container for holding the liquid; the dispensing mechanism throws the liquid in the container into the extraction column; and the pressurizing mechanism passes the liquid put into the extraction column through the nucleic acid-binding carrier. The nucleic acid extraction apparatus according to claim 8, wherein the nucleic acid extraction apparatus is discharged.   9. The nucleic acid recovery apparatus according to claim 8, wherein the nucleic acid-binding carrier is a substance containing silicon oxide.   The nucleic acid recovery apparatus according to claim 8, wherein the liquid is an alkaline solution.   The nucleic acid recovery apparatus according to claim 8, wherein the liquid contains an organic solvent.   The nucleic acid recovery apparatus according to claim 8, wherein the liquids are two or more liquids having different compositions, and the two or more liquids are sequentially brought into contact with the nucleic acid-binding carrier.   The nucleic acid recovery apparatus according to claim 8, wherein the nucleic acid is a ribonucleotide.

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