JP2014176393A - Simple and rapid nucleic acid extraction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for extracting a nucleic acid more simply and rapidly than a conventional method by targeting a nucleic acid-containing solution, especially a nucleic acid contained in an organism sample.SOLUTION: Both liquid passage for sucking and discharging a nucleic acid-containing solution under atmospheric pressure and nucleic acid recovery efficiency are satisfied by using a monolith structure having a restricted liquid passage hole, diameter and thickness as a nucleic acid adsorption carrier of a nucleic acid recovery container.

Description

The present invention relates to a method for conveniently and rapidly extracting a nucleic acid from a sample mixed with impurities.

In recent years, in the field of clinical testing, genetic testing targeting pathogenic bacterial genes and genes involved in drug metabolism has become widespread. Genetic testing is a test that is superior in sensitivity and speed compared to other clinical tests because it amplifies nucleic acids exponentially in a short time. Various techniques have been developed for nucleic acid amplification techniques. For example, PCR, NASBA, LCR, SDA, RCR, TMA, LAMP, ICAN and the like can be mentioned. These technologies are amplified using the nucleic acid contained in the sample as a template, and are susceptible to inhibition depending on the state of the sample before amplification. It is known that the pretreatment method is particularly important.

The technology that is the basis of the currently used nucleic acid extraction method is a method using a chaotropic agent and silica shown in Patent Document 1, which purifies nucleic acid from human serum or urine. it can. Specifically, a sample containing nucleic acid is added to a reaction vessel containing a suspension of silica particles and a guanidine thiocyanate buffer solution as a chaotropic substance, and mixed to form a complex in which the nucleic acid is adsorbed (bound) to the silica particles. After sedimentation by centrifugation, the supernatant is discarded, and the remaining complex is washed with a washing solution. The reprecipitated complex is washed with an aqueous ethanol solution, then washed with acetone, and the acetone is removed and dried. A method of eluting and recovering nucleic acids by adding an elution buffer to the complex is described.

Various methods that further develop the above method are disclosed. Patent Document 2 describes a method for easily extracting nucleic acids using magnetic particles. Magnetic particles that do not specifically adsorb nucleic acids are suspended in a solution containing nucleic acids, and salts and alcohol are added to insolubilize the nucleic acids, and the nucleic acids are aggregated around the magnetic particles by cooling. Apply a magnetic field to agglomerate the complex of magnetic particles and nucleic acid, remove the supernatant, add eluate to redissolve the nucleic acid, apply a magnetic field to precipitate the magnetic particles, and remove the nucleic acid-dissolved supernatant. obtain.

Patent Document 3 discloses a method for efficiently extracting nucleic acids using a column holding a silica carrier. A column is formed by inserting a net-like thin film made of silica gel, glass or quartz fiber having a pore diameter of <5 μm between polyethylene frit having a pore diameter of 50 to 200 μm held in a hollow body. Using the column, a solution containing nucleic acid is mixed with a buffer solution having a high ionic strength, and the mixed solution is adsorbed by passing through a thin film by suction or centrifugation. Further, it is washed with a washing solution containing alcohol and eluted with a low ionic strength buffer. Compared with the “batch type” method in which silica particles are used as a nucleic acid adsorption carrier, the efficiency of nucleic acid adsorption is increased because the liquid flows in one direction.

Patent Document 4 discloses a nucleic acid purification method using an integrated monolith structure as a nucleic acid adsorption carrier in a column. By using the monolith structure, there are advantages that there is little liquid residue and the centrifugal force is less than that of the nucleic acid purification method using a conventional column.

Patent Document 5 discloses a method for extracting nucleic acids more easily. A nucleic acid-capturing chip containing a silica-containing solid phase is detachably connected to a movable nozzle for sucking and discharging liquid, and a liquid mixture of a substance that promotes adsorption of nucleic acid to the solid phase and a nucleic acid-containing sample is discharged from a predetermined container. After sucking into the nucleic acid capture chip connected to the movable nozzle for adsorption and adsorbing the nucleic acid to the solid phase, the liquid in the nucleic acid capture chip is removed, and then the washing liquid is aspirated and discharged to evacuate the nucleic acid capture chip. After washing, the eluent is sucked into the washed nucleic acid capturing chip, and the eluent containing the nucleic acid eluted from the solid phase is discharged into the purified product container.

Japanese Patent No. 2680462 Patent No. 2703114 Patent Publication No. 8-501321 JP 2005-224167 A JP-A-11-266864

The nucleic acid extraction method using magnetic particles described above has an advantage that it can be easily automated, but nucleic acid amplification is inhibited by residual alcohol adhering to the aggregated particles and fine magnetic particles suspended in the nucleic acid dissolution supernatant. There was a problem.

In addition, the nucleic acid extraction method using a column holding a silica carrier is not a simple method because a special device such as a suction device or a centrifuge is required to pass the solution through the column.

The method using a nucleic acid capture chip containing a silica-containing solid phase does not require a special device such as a centrifuge. However, while pursuing simplicity, a sample containing a large amount of impurities and a decrease in nucleic acid recovery efficiency has been pursued. There is a problem that the carrier is clogged and the liquid cannot be sucked.

An object of the present invention is to provide a method for extracting a nucleic acid more easily and more quickly than before by using a nucleic acid-containing solution, particularly a nucleic acid contained in a biological sample as a target.

As a result of intensive studies in view of the above-mentioned problems, the present inventors sucked a nucleic acid-containing solution at a pressure lower than atmospheric pressure by using a monolith structure having a limited hole, diameter, and thickness as a nucleic acid adsorption carrier for a nucleic acid recovery container. -The present invention has been completed by finding out that both the dischargeable liquid and the nucleic acid recovery efficiency can be achieved. That is, the present invention has the following configuration.

[Claim 1]
A method for extracting nucleic acid from a solution containing nucleic acid,
In a method for extracting nucleic acid by specific adsorption / washing and elution of a nucleic acid on a solid phase carrier using a device having a mechanism capable of performing suction and discharge using pressure using air as a medium,
A nucleic acid extraction method comprising the following steps (1) to (6):
(1) A step of preparing a container having the following features (a) and (b).
(A) Open surfaces are provided in two directions: a side for sucking and discharging a solution containing a nucleic acid (liquid passing part side) and a side for adjusting pressure using air as a medium (venting part side).
(B) A monolith structure having an average pore diameter of 20 μm or more and 100 μm or less is fixed as a solid phase carrier so as to completely divide the space of the container into two.
(2) A step of connecting the vent portion side of the container to a suction / discharge mechanism.
(3) A step of mixing a solution containing nucleic acid and an adsorbent.
(4) The adsorbing liquid mixed with the solution containing the nucleic acid is sucked from the liquid passing part side of the container and brought into contact with the monolith structure to adsorb the nucleic acid to the monolith structure, and then the liquid in the container is discharged. Process.
(5) A step of sucking the cleaning liquid from the liquid passing part side of the container, bringing the cleaning liquid into contact with the monolith structure, cleaning the inside of the monolith structure and the inside of the container, and then discharging the liquid in the container.
(6) A step of aspirating the eluate from the liquid passage side of the container to elute the nucleic acid adsorbed on the monolith structure, and then discharging the liquid in the container.
[Section 2]
Item 2. The method for extracting nucleic acid according to Item 1, wherein a cut area in a direction perpendicular to the liquid passing direction of the monolith structure is 0.1 square mm or more and 100 square mm or less.
[Section 3]
Item 3. The method for extracting nucleic acid according to Item 1 or 2, wherein the monolith structure has a thickness in the liquid passing direction of 0.2 mm or more and 5 mm or less.
[Claim 4]
Item 4. The method for extracting nucleic acid according to any one of Items 1 to 3, wherein the solution suction speed is faster than 10 μL / second and slower than 500 μL / second.
[Section 5]
Item 5. The method for extracting nucleic acid according to any one of Items 1 to 4, wherein the solution discharge rate is faster than 10 μL / second and slower than 500 μL / second.
[Claim 6]
Item 2. The method for extracting nucleic acid according to Item 1, wherein the adsorbing solution contains a chaotropic salt.
[Claim 7]
Item 7. The nucleic acid extraction method according to Item 6, wherein the adsorbing solution contains a chaotropic salt and contains an acetate salt.
[Section 8]
Item 8. The nucleic acid extraction method according to any one of Items 6 and 7, wherein the chaotropic salt is at least one selected from the group consisting of a guanidine salt, an iodide salt and urea.
[Claim 9]
Item 2. The method for extracting nucleic acid according to Item 1, wherein the washing liquid contains alcohols.
[Section 10]
Item 10. The method for extracting nucleic acid according to Item 9, wherein the washing liquid contains alcohols and acetate.
[Section 11]
Item 11. The nucleic acid extraction method according to Item 9 or 10, wherein the alcohol is any one or more selected from the group consisting of ethanol, isopropanol, and 1-propanol.
[Claim 12]
Item 11. The nucleic acid extraction method according to Item 7 or 10, wherein the acetate salt is any one or more selected from the group consisting of sodium acetate, potassium acetate, ammonium acetate, and lithium acetate.
[Claim 13]
Item 2. The method for extracting nucleic acid according to Item 1, wherein the eluate is a solution having a pH of 7 or more.

According to the present invention, as described above, nucleic acid recovery efficiency is good, and nucleic acid can be extracted by a quick and simple operation. Specifically, nucleic acid extraction, which has been a complicated operation, can be realized with a cheaper and simpler operation. Also, automation can be easily achieved, and application to a smaller and less expensive device becomes possible.

An example of a chip to which a monolith structure is fixed Schematic of the method for adding and recovering nucleic acid using a monolith chip. Monolith tip suction method

One embodiment of the present invention is a method for extracting a nucleic acid from a solution containing the nucleic acid, and using a device having a mechanism capable of aspirating and discharging using air using air as a medium, to a solid phase nucleic acid carrier In a method for extracting nucleic acids by specific binding, washing and elution of
It is a nucleic acid extraction method including the following steps (1) to (6).
(1) A step of preparing a container having the following features (a) and (b).
(A) Open surfaces are provided in two directions: a side for sucking and discharging a solution containing a nucleic acid (liquid passing part side) and a side for adjusting pressure using air as a medium (venting part side).
(B) A monolith structure having an average pore diameter of 20 μm or more and 100 μm or less is fixed as a solid phase carrier so as to completely divide the space of the container into two.
(2) A step of connecting the vent portion side of the container to a suction / discharge mechanism.
(3) A step of mixing a solution containing nucleic acid and an adsorbent.
(4) The adsorbing liquid mixed with the solution containing the nucleic acid is sucked from the liquid passing part side of the container and brought into contact with the monolith structure to adsorb the nucleic acid to the monolith structure, and then the liquid in the container is discharged. Process.
(5) A step of sucking the cleaning liquid from the liquid passing part side of the container, bringing the cleaning liquid into contact with the monolith structure, cleaning the inside of the monolith structure and the inside of the container, and then discharging the liquid in the container.
(6) A step of aspirating the eluate from the liquid passage side of the container to elute the nucleic acid adsorbed on the monolith structure, and then discharging the liquid in the container.

  One of the features in the embodiment of the present invention is that a monolith structure having an open surface in two directions of the liquid passing part side and the venting part side and having an average pore diameter of 20 μm or more and 100 μm or less as a solid phase carrier, It is to use a container which is fixed so that the space of the container is completely divided into two.

[Production method of monolith structure]
The monolith structure used in the present invention means an integrally molded porous body and can be produced by the method exemplified below, but is not particularly limited.

The monolith structure can be mainly produced by a sol-gel method. That is, a metal alkoxide is partially hydrolyzed to produce a reactive monomer, and this monomer is polycondensed to form a colloidal oligomer (formation of a sol), and further hydrolyzed to promote polymerization and cross-linking, thereby providing a three-dimensional It is synthesized by creating a structure (generation of gel).
When various organic monomers are added during this reaction, an organic / inorganic hybrid monolith can be easily prepared. Therefore, chemical characteristics can be added depending on the type of organic monomer to be added.
For example, the water absorption of the aqueous sample component can be improved by adding an organic monomer having a hydrophilic group. In addition, by mixing organic and inorganic, it is possible to increase the chemical stability of the monolith structure. The organic / inorganic hybrid monolith structure produced by the sol-gel method can add properties such as constituting a chemical surface according to the purpose and improving chemical stability depending on the kind of the added organic monomer. It shows that the monolith properties effective in nucleic acid extraction can be freely improved according to the purpose.

Furthermore, the monolith structure produced from the sol-gel method is suitable in that it contains less metal. General silica gel or the like is made from sodium silicate or the like, and a large amount of metal remains. Certainly, there are some high-purity silica gels made from purified sodium silicate and sol-gel methods, but they are expensive and are not suitable as a nucleic acid recovery container for disposable use. Even if it can be made inexpensively, it is batch-type synthesis at the time of particle preparation, and there is a high possibility that metal concentration will occur from the synthesis atmosphere. In the synthesis of the monolith structure in the sol-gel method, it is produced by a continuous process, and there is no metal contamination. Conventionally, silica gel has been devised such as washing with hydrochloric acid, nitric acid or the like, or addition of EDTA or the like to reduce the metal effect, but the monolith structure of the present invention is not necessary at all.

Alternatively, monolith structures can be created by glass phase separation.
Basically, it is as effective as the synthesis of the monolith structure from the sol-gel method,
Since macropores can be made larger than gel monoliths, it is effective when secondary micropores are formed on the inner surface. Further, the glass phase separation has the advantage that it has higher alkali resistance than its composition and can be regenerated by alkali cleaning.

It is generally known that the monolith structure produced by the sol-gel method as described above is used in, for example, an HPLC column.

Silanol groups are imparted to the silica surface by the method described above.
The monolith structure used for nucleic acid extraction preferably has a silanol group on the surface in order to adsorb and desorb nucleic acids on the same principle as the nucleic acid extraction method using silica, which is widely known as the boom method.

[Characteristics of monolith structure]
One of the features of the embodiment of the present invention is that a monolith structure having an average pore diameter of 20 μm or more and 100 μm or less is used.

  The advantage of the monolith structure is that it can be produced by controlling the pore diameter through which the liquid passes. In the present invention, since the solution is passed by sucking and discharging the solution, the liquid clogging occurs at the pore diameter of the monolith structure that has been conventionally allowed to pass the solution by centrifugation. The pore diameter of the monolith structure suitable for suction / discharge is not limited as long as the solution is not clogged by suction / discharge and the nucleic acid can be recovered.

  When sucking and discharging a solution by a pressure change, the viscosity and purity of the solution are related to clogging. In particular, in the case of a biological sample, since there are many impurities such as proteins, there is a high possibility of causing liquid clogging. The pore size of the monolith structure suitable for nucleic acid extraction from such a biological sample is preferably 20 μm or more. Further, in view of liquid permeability, when a highly viscous solution is used, 30 μm or more is more preferable. When the thickness is less than 20 μm, it is difficult to pass the monolith structure when impurities are contained in the used solution in the suction / discharge operation under atmospheric pressure.

Further, the adsorption of the nucleic acid to the monolith structure requires contact between the nucleic acid in the solution and the monolith structure. In order for the nucleic acid to efficiently contact the surface of the monolith structure, the pore diameter through which the solution passes is preferably small, and is not particularly limited, but is preferably 100 μm or less, more preferably 60 μm or less, further preferably 50 μm or less, and more preferably 40 μm or less. Further preferred.

The lower limit of the pore diameter of the monolith structure that achieves both liquid permeability and nucleic acid adsorption efficiency is preferably 20 μm or more, and more preferably 30 μm. The upper limit is preferably 60 μm or less, and more preferably 40 μm or less. The pore diameter of such a monolith structure is determined from the average diameter of the liquid passage holes from an electron microscope image. For more accurate measurement, the mercury intrusion method can be used.

The volume of the monolith structure also affects liquid permeability and nucleic acid adsorption efficiency.
At the time of suction and discharge of the adsorbing liquid and the cleaning liquid, the volume of the solution is much larger than the volume of the monolith structure, so that the passage of the solution is not affected. However, in the case of an eluate, it is preferable to use a small amount as much as possible for concentration of the nucleic acid, and in the case of a small amount, it becomes difficult to control the solution passage speed when passing through the monolith structure. When the volume of the eluate is the same as the volume of the monolith structure, it is highly possible that the eluate contains gas when sucked and discharged, and it is extremely difficult to control the eluate. For example, the volume ratio of the eluate to be used and the monolith structure is preferably 2 or more. More preferably, it is 4 or more. More preferably, it is 5 or more. Although not particularly limited, the shape of the monolith structure may be a cylindrical shape, a rectangular parallelepiped, a conical shape, a shape obtained by cutting a conical shape, a sphere, or the like. The shape is selected depending on the shape and size of the container.

  In the present invention, the cut area of the monolith structure is preferably 0.1 square mm to 100 square mm, and more preferably 1 square mm to 30 square mm. More preferably, they are 1 square mm or more and 50 square mm or less, and are 3 square mm or more and 20 square mm or less.

  The cut area in the direction perpendicular to the liquid passing direction of the monolith structure indicates the area of the surface of the monolith structure cut parallel to the open surface when the monolith structure is fixed in the container. The cutting position is defined as the area of the surface cut in the middle of the monolith structure. The shape of the cut surface is mainly circular or square, but is not limited thereto.

In order to perform welding between the monolith structure and the container to be fixed without a gap, the cut surface is preferably circular, and the cut area can be calculated by the product of radius, radius, and 3.14.

The liquid permeability and nucleic acid adsorption efficiency of the monolith structure vary depending on the shape even in the same volume. For example, when a cylindrical monolith structure is used, the diameter is preferably 0.2 mm or more and the thickness in the liquid passing direction is 0.2 mm or more from the viewpoint of manufacturing the monolith structure or ensuring the strength. The thickness is preferably 0.2 mm or more and less than 5 mm, and more preferably 0.4 mm or more and less than 4 mm. More preferably, it is 0.8 mm or more and less than 2 mm. If the thickness is increased, the resistance generated in the monolith structure increases, and it becomes difficult to pass liquid. The diameter is preferably 0.2 mm or more and less than 10 mm, and more preferably 0.5 mm or more and less than 8 mm. More preferably, it is 1 mm or more and less than 5 mm. When the thickness exceeds 10 mm, the solution resistance due to the monolith structure increases, and it becomes difficult to suck and discharge the solution. If suction and discharge forces are increased to perform suction and discharge, nucleic acid recovery efficiency is reduced due to the generation of bubbles. When the diameter or thickness is less than 0.2 mm, the strength of the monolith structure is insufficient and fragile and is not suitable for the shape of the monolith structure of the present invention.

[container]
In the present invention, the monolith structure as described above is provided with open surfaces in two directions: a side for sucking and discharging a solution containing nucleic acid (liquid passing part side) and a side for adjusting pressure using air as a medium (venting part side). Secure to the container that holds it. In the container, the monolith structure is fixed so that the space of the container is completely divided into two.
The position where the monolith structure is fixed to the container is not particularly limited, but it is preferable that the monolith structure is on the liquid passing side from the center of the container. It is more preferable that the distance from the monolith structure to the open surface on the liquid passage side is within 20 mm. The distance from the monolith structure to the open surface on the liquid passage side causes an increase in the remaining solution after the solution is discharged. For this reason, the distance from the monolith structure to the open surface on the liquid passing part side is preferably as short as possible in view of the structure of the container for sucking and discharging the solution.

A pipette tip is suitable for the container for fixing the monolith structure. The shape of the tip is not particularly limited, but a tip on a cylinder is preferable because the tip is cut in a generally used conical shape. For example, a chip of 10 μL to 5000 μL manufactured by Rainin, or a chip of 10 μL to 5000 μL manufactured by Eppendorf can be used. An example in which a monolith structure is fixed to a pipette tip is shown in FIG. Further, the shape is not limited to the pipette tip, and any shape can be selected as long as the shape can be in close contact with a mating portion with a device that causes a pressure change.
The monolith structure may be fixed to the chip as long as the chip and the monolith structure are firmly fixed. For example, the monolith structure can be fixed by an ultrasonic welding method.

[Solution containing nucleic acid]
The solution containing the nucleic acid is not particularly limited, and examples thereof include biological samples such as blood, serum, blood cells, sputum, urine, gastric juice, and swab. Samples subjected to a pretreatment method suitable for each biological sample are also included. In addition, cultured tissues, cells, bacteria, viruses and the like can also be mentioned. Furthermore, this also applies to a solution obtained by extracting nucleic acid by an optimum method or a purified nucleic acid.

[Apparatus for sucking and discharging]
As a device having a mechanism capable of performing suction and discharge using pressure using air as a medium, that is, a device for applying a pressure change to a container to which a monolith structure is fixed (also referred to as “suction and discharge device”), suction and discharge of a solution Although it will not specifically limit if it can do, The apparatus which can control a pressure change is preferable. For example, a pipette or a syringe that can be operated manually can be used. In addition, an automatic device that fits a pipette tip like an automatic dispenser and can accurately control the liquid amount and speed during suction and discharge is also suitable. For example, using a Multi-CHOT (registered trademark) 1000 μL disposable tip head manufactured by Nichiyo, a container to which a monolith structure is fixed can be fitted and suction / discharge can be performed like a normal pipette tip.

It is desirable that air does not leak at the mating portion between the container in which the monolith structure is fixed and the device that generates the pressure change of the suction and discharge of the solution. When air leaks at the joint between the container and the apparatus, the suction and discharge forces are significantly reduced, and the passage of the solution through the monolith structure cannot be controlled.

The liquid passing portion in the container indicates a portion that comes into contact with the solution, and is a portion through which the solution first passes when sucked. The liquid passage part has a shape that facilitates suction of the solution, and a shape like the tip of a pipette tip is preferable. In addition, the ventilation portion indicates an open surface to which a device for generating a pressure change can be connected, and is a portion through which gas passes first when suctioning. Depending on the amount of adsorbing liquid or cleaning liquid, the vent may pass through the solution during suction and discharge.

[Solution suction speed]
In order to obtain a high nucleic acid recovery rate, it is necessary to increase the contact time between the nucleic acid in the solution and the surface of the monolith structure. In order to lengthen the contact time, it is preferable that the solution suction and discharge speed be as slow as possible. However, a simple and rapid nucleic acid extraction method is desired, and suction and discharge speeds that can achieve a high recovery rate in a short time are preferable.
The suction and discharge speeds are defined by the time (seconds) required to suck a fixed amount of solution or the time (seconds) required to discharge a fixed amount of solution. For example, when 500 μL of solution is sucked, the time from when the solution is sucked to the tip of the container with the monolith structure fixed to when 500 μL of the solution is all sucked into the container with the monolith structure fixed is measured. When it takes 2 seconds, it is set to 100 μL / second. Similarly, the discharge can be calculated by measuring the time at which 500 μL of the solution held inside the container holding the monolith structure is pushed out of the container holding the monolith structure.

  When the solution is aspirated rapidly, bubbles are generated in the solution containing the nucleic acid. Due to the generation of bubbles, the adsorption action of nucleic acid to the monolith structure is weakened and the contact time is shortened, so that the nucleic acid adsorption efficiency is lowered. A suction speed exceeding 500 μL / sec is difficult to achieve due to the resistance of the monolith structure. Although the upper limit of the suction speed varies depending on the viscosity of the solution, it is not limited. However, if the solution can be sucked by a general pipetting operation, the suction speed is preferably 500 μL / second or less, and more preferably 300 μL / second or less. 200 μL / second or less is more preferable.

When the solution is rapidly discharged, the nucleic acid adsorbed on the monolith structure may be physically desorbed. Nucleic acid desorption during ejection requires adsorption to the monolith structure again, which can result in loss of adsorption efficiency. A discharge speed exceeding 500 μL / second is difficult to achieve due to the resistance of the monolith structure. Since the upper limit of the discharge speed differs depending on the viscosity of the solution as well as the suction speed, it is not limited. However, if the solution can be discharged by a general pipetting operation, for example, 500 μL / second or less is preferable, and 300 μL / second or less is more preferable. More preferably, it is 200 μL / second or less.
The lower limit of the suction / discharge speed is not particularly limited as long as it is appropriately set to such an extent that the rapidity is not impaired in consideration of the entire processing time.

[Nucleic acid extraction method]
In the nucleic acid extraction method of the present invention, the vent portion side of the container to which the monolith structure is fixed is connected to a suction / discharge device, and the same principle as the nucleic acid extraction method using silica, which is widely known as a boom method, is used. The nucleic acid is extracted by specific binding, washing and elution of the nucleic acid to the solid phase carrier.

[Binding of nucleic acid to monolith structure]
Specific binding of the nucleic acid to the solid phase carrier is performed by mixing a solution containing the nucleic acid with the adsorbing liquid, sucking it from the liquid passing part side of the container, and bringing it into contact with the monolith structure.
The adsorbing liquid preferably has a composition that can efficiently adsorb nucleic acid to the surface of the monolith structure.
Furthermore, a solution having low viscosity and less foaming is suitable. Although not particularly limited, a chaotropic salt used when adsorbing a nucleic acid on a silica support can be used. Examples thereof include guanidinium salts and iodide salts. In particular, the guanidinium salt is preferably used in a concentration range of 1M to 8M. More specifically, guanidine hydrochloride, guanidine thiocyanate, guanidine nitrate, guanidine sulfate and the like can be used as the guanidinium salt. Moreover, sodium iodide, potassium iodide, etc. can be used as an iodide salt. In addition, although sodium thiocyanate, urea, etc. can be used, it is not restricted to this.
The pH of the adsorbing liquid is preferably 2-7. More preferably, the pH is 3-6. A buffer solution can be used to stabilize the pH of the adsorbed solution. A salt such as acetate can also be added. The acetate is preferably a salt with a monovalent anion. For example, sodium acetate, potassium acetate, ammonium acetate, or lithium acetate can be used.

[Washing]
After the nucleic acid is specifically bound to the monolith structure, the liquid in the container is discharged, and then the monolith structure and the container are washed.
The cleaning is performed by sucking the cleaning liquid from the liquid passing part side of the container, bringing the cleaning liquid into contact with the monolith structure, cleaning the inside of the monolith structure and the inside of the container, and then discharging the liquid in the container.
The washing solution is preferably a solution that does not desorb the nucleic acid adsorbed on the monolith structure. Furthermore, a solution having low viscosity and less foaming is suitable. Examples include solutions that do not dissolve nucleic acids, such as alcohols having a concentration of 30% or more. The alcohol is preferably ethanol, isopropanol, or 1-propanol. A combination of alcohols and chaotropic salts is also suitable as a reagent composition for the cleaning liquid. The chaotropic salt may be a concentration lower than the concentration of the chaotropic salt in the adsorbing solution, preferably 3M or less, more preferably 2M or less, and even more preferably 1M or less. When the alcohols are used by mixing with a chaotropic salt, the concentration is preferably 10% or more, and more preferably 30% or more.

[Elution]
After washing, the nucleic acid is eluted from the monolith structure.
The elution is performed by sucking the eluate from the liquid passage side of the container, bringing the eluate into contact with the monolith structure to elute the nucleic acid adsorbed on the monolith structure, and then discharging the liquid in the container.
The eluate is preferably a solution that can dissolve nucleic acids and desorb from the monolith structure. Furthermore, a solution composition that does not inhibit a reaction after nucleic acid extraction, for example, a nucleic acid amplification reaction typified by PCR is preferable. For example, a low-concentration buffer solution such as a Tris buffer solution, a solution having a pH of 7 or more such as a sodium hydroxide solution or a potassium hydroxide solution, preferably an alkaline solution. Further, the nucleic acid adsorbed on the monolith structure can be eluted by raising the temperature of the eluate. In this case, it is possible to elute the adsorbed nucleic acid efficiently without using an alkaline solution.

[Number of suction and discharge]
In each of the above binding, washing, and elution steps, suction and discharge may be performed a plurality of times.
In the binding (adsorption) step, the contact time between the nucleic acid and the monolith structure can be extended by increasing the number of times the solution containing the nucleic acid is sucked and discharged, and this is in parallel with the suction / discharge speed. The adsorption efficiency is affected. Although it is preferable to increase the number of times in terms of increasing the contact opportunity, it is desirable to obtain a high adsorption efficiency with a smaller number of times in order to achieve rapid nucleic acid extraction as well as speed.

  In order to adsorb the nucleic acid to the monolith structure in an almost 100% state, the number of times of the adsorbing liquid is more important, preferably at least 2 times, and more preferably 3 times. Further, it is preferably 5 times or more, more preferably 10 times or more. For example, when suction / discharge is performed at a speed of 100 μL / second, nucleic acid adsorption is almost 100% if the adsorption liquid is sucked / discharged 10 times.

  In the washing process, the number of suction / discharge is not limited because the washing liquid is intended to remove and dilute the adsorbing liquid without desorbing nucleic acids. Rather, it is preferable to use a new cleaning solution that is not used for cleaning at least twice. More preferably, the washing step is repeated three times or more.

In the elution step, the number of times the eluate is sucked and discharged is preferably such that the nucleic acid adsorbed on the monolith structure can be dissolved nearly 100%. Since the remaining of the washing solution prevents the nucleic acid from being dissolved in the eluate, at least two suction / discharge cycles are preferred. At least 3 times or more is more preferable, and at least 5 times or more is more preferable. If the suction and discharge are repeated five times or more, the remaining washing solution dissolves in the eluate, and the nucleic acid adsorbed on the monolith structure comes into contact with the eluate, so that the nucleic acid does not remain in the monolith structure. Is eluted.

[Addition and recovery of bacterial genomes using monolith structures with different pore sizes]
(1) Preparation of sample As a bacterial genome, the genome of Mycobacterium bovis BCG strain was used. After culturing in 3% Ogawa medium (manufactured by Nissui Pharmaceutical Co., Ltd.), genomic DNA was extracted by the phenol / chloroform method to obtain a sample for addition recovery.

(2) Preparation of material A monolith structure having a pore diameter different from 10 μm, 20 μm, 30 μm, 40 μm, and 50 μm was fixed to a Rainin LTS 1000 μL pipette tip by ultrasonic welding (hereinafter abbreviated as a monolith chip). . Other properties of the monolith structure were all set to a cutting area of 3.14 square mm and a thickness of 1 mm. A Terumo 1 mL syringe was used as a device for causing a pressure change. The adsorbing solution was 5M guanidine thiocyanic acid, 0.8M potassium acetate solution, the washing solution was 0.8M potassium acetate solution, 70% ethanol, and the eluent was 10 mM potassium hydroxide solution. As the sample, the extracted sample for addition collection was diluted with 10 mM Tris buffer so as to contain about 1000 copies in 1 μL.

(3) Addition and collection of bacterial genome with monolith chip Using the monolith chip of 5 types having different pore diameters of the monolith structure, operations as shown in the simplified diagram of FIG. 2 were performed.
Details are described below. 500 μL of the adsorbing solution was dispensed into a 1.5 mL tube, 100 μL of the sample was added, and mixed 5 times by pipetting. Using each monolith chip attached to the syringe, the adsorbed solution / sample mixed solution was sucked and discharged 10 times at a rate of 100 μL / second. After the mixture was completely discharged, the monolith chip was moved to a tube containing 500 μL of cleaning solution, and the cleaning solution was suctioned and discharged three times at a rate of 100 μL / second for cleaning. The washing process was repeated once more. Finally, the monolith chip was moved to a tube containing 100 μL of the eluate, and the eluate was aspirated and discharged 5 times at a speed of 100 μL / sec to elute the genomic DNA adsorbed in the monolith chip. In order to suppress a decrease in elution efficiency, care was taken so that air did not contact the monolith structure during suction and discharge.

(4) 5 types of genomic DNA solutions added and recovered using monolith chips with different pore diameters of the real-time PCR detection monolith structure, samples (standard substances) not subjected to addition and recovery using the monolith chips, and negative controls In addition to the reagent shown in (5), real-time PCR detection was performed under the conditions shown in (6). The reagent using Oligo 1 and Oligo 2 as primers and Oligo 3 as a probe is a combination of a primer and a probe that can specifically detect the BCG strain. The detection principle is that the fluorescence of the fluorescent dye labeled on the probe is quenched by hybridization of the amplification product and the probe. A light cycler (registered trademark) manufactured by Roche Diagnostics was used for nucleic acid amplification and detection. The measurement mode was 530 nm, and the Q Probe extinction rate during annealing was calculated using the obtained real-time detection data. Further, the number of cycles that reached a quenching rate of 2% was determined and analyzed in the same manner as the conventional real-time quantitative PCR method using SYBR Green I. The addition recovery efficiency was calculated for each sample with the detection of the standard substance as 100%.

(5) Reagent composition A 10 μL solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 1 (SEQ ID NO: 1) 250 nM,
Oligo 2 (SEQ ID NO: 2) 1500 nM,
Oligo 3 (SEQ ID NO: 3) (5 ′ end labeled with BODIPY-FL) 250 nM,
× 10 buffer 1 μL,
dNTP 0.2 mM,
MgSO ₄ 4 mM,
KOD plus DNA polymerase 0.2U,
Eluate 1μL

(6) Nucleic acid amplification and detection conditions 94 ° C 2 minutes 98 ° C 0 seconds, 60 ° C 5 seconds (fluorescence detection), 50 cycles 94 ° C 60 seconds, 40 ° C 60 seconds, 40 ° C to 75 ° C 0.5 ° C / second Temperature rise

(7) Results Table 1 shows addition recovery rates detected and analyzed using genomic DNA solutions added and recovered using five types of monolith chips having different pore sizes of monolith structures. As can be seen from Table 1, the monolith structure could be recovered when the pore diameter was 20 μm to 50 μm. From 20 μm to 40 μm, the recovery rate was 50% or more, and good results were shown.
When recovery was performed using a monolith structure having a pore diameter of 10 μm, it was difficult to suck and discharge the adsorbed liquid due to clogging, and the addition and recovery operation was abandoned.

[Addition and recovery of bacterial genome using monolith chips with different diameters]
(1) Used material, operation method, measurement method, analysis method A monolith structure having a diameter different from 0.4 mm, 1.5 mm, and 2 mm is fixed to a Rainin LTS 1000 μL pipette tip (hereinafter abbreviated as a monolith tip). did. In addition, those having a diameter of 4 mm and 10 mm were fixed to containers having an inner diameter of 4 mm and 10 mm, respectively, which can be fitted with a 1 mL syringe. Other properties of the monolith structure were all set to an average pore diameter of 30 μm and a thickness of 1 mm. The respective cut areas are 0.126 square mm, 1.767 square mm, 3.14 square mm, and 78.5 square mm. Other materials, operation methods, measurement methods, and analysis methods were carried out according to Example 1.

(2) Results Table 2 shows addition recovery rates detected and analyzed using genomic DNA solutions added and recovered using five types of monolith chips having different cut areas of the monolith structure. As is apparent from Table 2, nucleic acid could be recovered when the diameter of the monolith structure was 0.4 mm to 10 mm. When the diameter was 10 mm, the amount of residual liquid in the monolith structure increased and the amount of solution after elution decreased. Also, if the suction and discharge speed is 200 μL / second or more, air tends to enter during the elution process, so care must be taken.

[Addition and recovery of bacterial genome using monolith chips of different thickness]
(1) Used material, operation method, measurement method, analysis method A monolith structure having a thickness different from 1 mm, 2 mm, 5 mm, and 10 mm was fixed to a Rainin LTS 1000 μL pipette tip (hereinafter abbreviated as a monolith tip). . The properties of the other monolith structures were all set to an average pore diameter of 30 μm and a cut surface of 3.14 square mm. Other materials, operation methods, measurement methods, and analysis methods were carried out according to Example 1.

(2) Results Table 3 shows addition recovery rates detected and analyzed using genomic DNA solutions added and recovered using four types of monolith chips having different monolith structure thicknesses. As is clear from Table 3, the nucleic acid could be recovered when the monolith structure had a thickness of 1 mm to 5 mm. When the thickness was 10 mm, the flow resistance of the monolith structure increased, and it took 10 minutes or more for the operation, which was not suitable for rapid nucleic acid extraction.

[Liquid permeability test of monolith chip]
(1) Preparation of material The monolith structure was cylindrical and had a basic structure with an average pore diameter of 30 μm, a diameter of 2 mm, and a thickness of 1 mm. Average pore diameter changed to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, diameter changed to 0.4 mm, 1.5 mm, 2 mm, 4 mm, 10 mm, thickness changed to 1 mm, 2 mm, 5 mm, 10 mm A total of 14 types were used. A monolith chip was prepared by fixing each to a suitable container. As the viscous solution used for the test, the adsorption solution of Example 1 contained so as to be a 0.1% mucin solution (manufactured by Sigma) was used. As a device for causing a pressure change, a 1 mL syringe was used as in Example 1.

(2) Liquid permeability test method 100 μL of the viscous solution was mixed with 500 μL of the adsorbing solution dispensed in the 1.5 mL tube, and mixed well by pipetting. Using the monolith chip to be tested, a total volume of 600 μL is aspirated (with no viscous solution in the tube and the bottom surface of the liquid is under the monolith structure, illustrated in FIG. 3), and then discharged in a full volume (monolith) The time until the solution in the chip was completely discharged) was measured.

(3) Criteria for determination of liquid permeability test The liquid permeability was good when the time taken when sucking and discharging using the viscous solution was within 60 seconds. When it exceeded 60 seconds, it was considered as poor liquid permeability.

(4) Results As shown in Tables 4 to 6, in the case of using a monolith structure that was changed to an average pore diameter of 10 μm, 20 μm, a diameter of 0.4 mm, a thickness of 5 mm, and 10 mm, respectively, Resistance was strong and liquid permeability was poor. In the case of a monolith structure that has been changed to an average pore size of 20 μm, a diameter of 0.4 mm, and a thickness of 5 mm, there is no problem as long as it is a genomic solution, but it is clogged when impurities such as clinical specimens are included and a viscous solution is used. There is a possibility.

[Comparison of addition and collection time of bacterial genome]
(1) Used material, operation method, measurement method, analysis method A monolith chip was prepared by fixing a monolith structure having an average pore diameter of 30 μm, a cut surface of 3.14 square mm, and a thickness of 1 mm to a Rainin LTS 1000 μL pipette tip. Other materials, operation methods, measurement methods, and analysis methods were carried out according to Example 1. It was carried out 3 times and each time was measured.

(2) Results The time from the mixing of the bacterial genome solution to the completion of elution was 4 minutes 00 seconds, 3 minutes 47 seconds, and 3 minutes 53 seconds. In Japanese Patent Application Laid-Open No. 2007-306967, which is disclosed as a nucleic acid purification method using suction / discharge, the time from mixing of a whole blood sample to the completion of elution is 16 minutes in the comparative example. The simultaneous method takes 8 minutes, and the method according to the present invention was found to be a very rapid nucleic acid purification method.

According to the present invention, it is possible to extract or purify a nucleic acid quickly and easily from a biological sample important for clinical diagnosis. Specifically, a nucleic acid can be extracted and purified within 10 minutes from a biological sample containing a gene amplification method inhibitor obtained by diagnosis. In addition, since the operation is only aspiration and discharge of the solution, the centrifugal operation is unnecessary and application to the apparatus is easily achieved. Therefore, the demand in the clinical field is high, and it is expected to make a significant contribution to the industry.

Claims (12)

A method for extracting deoxyribonucleic acid from a genomic DNA solution containing deoxyribonucleic acid, using a device having a mechanism capable of suctioning and discharging using pressure using air as a medium, and deoxyribonucleic acid specific to a solid phase carrier In the method of extracting deoxyribonucleic acid by adsorption, washing and elution,
A deoxyribonucleic acid extraction method comprising the following steps (1) to (6):
(1) A step of preparing a container having the following features (a) and (b).
(A) Open surfaces are provided in two directions: a side for sucking and discharging a solution containing deoxyribonucleic acid (liquid passing part side) and a side for adjusting pressure using air as a medium (venting part side).
(B) As a solid phase carrier, a monolith structure satisfying all of the following (A) to (C) is fixed so as to completely divide the space of the container into two.
(A) The average pore diameter is 20 μm or more and 30 μm or less. (B) The cut surface is circular and the diameter of the cut surface in the direction perpendicular to the liquid passing direction is 2.0 mm or more and 4.0 mm or less. (2) A step of connecting the vent portion side of the container to a suction / discharge mechanism.
(3) A step of mixing a solution containing deoxyribonucleic acid and an adsorbent.
(4) The adsorbed liquid mixed with the solution containing deoxyribonucleic acid is sucked from the liquid passage side of the container and brought into contact with the monolith structure to adsorb deoxyribonucleic acid to the monolith structure. Discharging step.
(5) A step of sucking the cleaning liquid from the liquid passing part side of the container, bringing the cleaning liquid into contact with the monolith structure, cleaning the inside of the monolith structure and the inside of the container, and then discharging the liquid in the container.
(6) A step of aspirating the eluate from the liquid passage side of the container to elute deoxyribonucleic acid adsorbed on the monolith structure, and then discharging the liquid in the container. 2. The deoxyribonucleic acid extraction method according to claim 1, wherein a cutting area in a direction perpendicular to the liquid passing direction of the monolith structure is 0.1 square mm or more and 100 square mm or less. The method for extracting deoxyribonucleic acid according to claim 1 or 2, wherein the solution suction rate is faster than 10 µL / sec and slower than 500 µL / sec. The method for extracting deoxyribonucleic acid according to any one of claims 1 to 3, wherein the solution discharge rate is faster than 10 µL / sec and slower than 500 µL / sec. The deoxyribonucleic acid extraction method according to claim 1, wherein the adsorbing solution contains a chaotropic salt. The deoxyribonucleic acid extraction method according to claim 5, wherein the adsorbing solution contains a chaotropic salt and contains an acetate salt. The deoxyribonucleic acid extraction method according to claim 5 or 6, wherein the chaotropic salt is at least one selected from the group consisting of a guanidine salt, an iodide salt and urea.   The deoxyribonucleic acid extraction method according to claim 1, wherein the washing solution contains alcohols.   The deoxyribonucleic acid extraction method according to claim 8, wherein the washing solution contains alcohols and acetate.   The method for extracting deoxyribonucleic acid according to claim 8 or 9, wherein the alcohol is any one or more selected from the group consisting of ethanol, isopropanol and 1-propanol. The method for extracting deoxyribonucleic acid according to claim 6 or 9, wherein the acetate is at least one selected from the group consisting of sodium acetate, potassium acetate, ammonium acetate and lithium acetate.   2. The method for extracting deoxyribonucleic acid according to claim 1, wherein the eluate is a solution having a pH of 7 or more.