JP3582632B2 - Container for nucleic acid extraction - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to containers used for extracting nucleic acids from various samples containing nucleic acids, particularly from biological samples.
[0002]
[Prior art]
BACKGROUND ART In many fields in recent years, nucleic acids have been actively extracted from biological samples and the like. For example, in genetic engineering or production of a DNA probe, an operation of extracting DNA or RNA from a cell that produces a target protein is performed. Further, in clinical diagnosis for detecting viral DNA or viral RNA using a DNA probe, an operation of extracting DNA or viral RNA to be detected from a biological sample is performed.
[0003]
Such an operation of extracting nucleic acids is very important in various fields. Conventionally, many nucleic acid extraction methods are known, such as the protease K method and phenol / chloroform extraction (for example, Gene Medicine Vol. 1, No. 1, p112-119, 1997).
[0004]
Japanese Patent Application Laid-Open No. 9-19292 also proposes a nucleic acid extraction method using a magnetic carrier. The magnetic carrier has such a property that when the magnet is brought close to the magnet, the whole particles are magnetized and attracted to the magnet, and when the magnet is released, the magnet immediately loses its magnetism and returns to its original state. If nucleic acids can be adsorbed on the surface of the magnetic carrier, the magnetic carrier can be efficiently collected and collected on the inner wall of the container when the magnetic carrier is washed by using a magnet. The method of separating solids and liquids using magnetic carriers has been developed as a method that replaces the conventionally used centrifugation method, not only for nucleic acid extraction but also for protein analysis and testing in immunodiagnosis, and has been put to practical use. It is.
[0005]
Containers used for these extraction operations include, for example, a so-called microtube with a lid made of plastic, as described in Japanese Patent Publication No. 4-31945. In this container, the lid is open at the time of injecting and discharging the reagent.On the other hand, in the reaction process of heating the liquid in the reaction tank at a certain temperature for a certain time, or in the process of performing centrifugation, The lid is used in a closed state.
[0006]
Further, Japanese Patent Application Laid-Open No. 8-43400 proposes a reaction vessel used for an analysis method utilizing a specific binding reaction. This has the feature of devising that the bottom of the container is inclined to promote the specific binding reaction, and it can be applied to nucleic acid extraction.However, in the reaction process, the container is used without a lid. It is structured to be.
[0007]
In addition, there are many containers for diagnosis (JP-A-4-320962), reaction containers for immunoassay (JP-A-4-363661), and reaction containers used for chemical analyzers (JP-B-63-20172). Containers have been proposed. However, most of them have a structure in which the lid of the container is opened when the liquid is poured into the container or when the liquid in the container is discharged.
[0008]
[Problems to be solved by the invention]
The nucleic acid extraction operation usually comprises erythrocyte hemolysis, leukocyte enrichment, cell membrane degradation and nucleic acid purification. The operations common to the above-mentioned extraction methods can be summarized in a block diagram. As shown in FIG. 1, a liquid injection operation, a mixing operation for a liquid reaction, a B / F separation (solid phase separation), and a removal of an unnecessary component liquid are performed. Alternatively, it consists of nucleic acid recovery.
[0009]
By the way, in clinical diagnosis, when extracting nucleic acids, a plurality of target specimens may be processed simultaneously. Since one extraction container is used for one sample, a large number of extraction containers are generally used in an operation in a clean bench or in an automatic nucleic acid extraction apparatus.
[0010]
Therefore, when a plurality of extraction containers are arranged in a limited space and the extraction operation is performed at the same time, for example, in operations such as injection and discharge of a sample or a reagent, a container with an open lid as described above is used. This raises the most critical issues in clinical diagnosis, such as infection due to the scattering of dangerous viruses contained in biological samples such as blood, and contamination due to carryover that occurs between containers. There is fear.
[0011]
Furthermore, in the conventional container, the tip of the tip is inserted into the container by a pipette operation for injecting and discharging the liquid, so that there is a risk that a factor that inhibits the reaction may be mixed into the container from the outside.
[0012]
The present invention has been made in order to solve the above-mentioned problems occurring in an open extraction container often used in a conventional nucleic acid extraction operation, and is intended to prevent contamination due to carryover. It is an object of the present invention to provide a container for extracting nucleic acid.
[0013]
[Means for Solving the Problems]
The present inventors have, as a result of intensive studies, to achieve the above object, have found that it is possible to prevent contamination due to carryover by using a closed container structure for nucleic acid extraction, The present invention has been reached.
[0014]
That is, the present invention is characterized in that a reaction tank portion for mixing and reacting a biological sample and a reagent, a drain cup portion for storing an unnecessary component liquid, and a tube for recovering nucleic acid are fixed to the same support plate. Is a container for nucleic acid extraction.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is characterized in that a reaction tank portion for mixing and reacting biological samples and reagents, a drain cup portion for storing unnecessary component liquid, and a tube for recovering nucleic acid are fixed to the same support plate. It is. It is preferable in terms of operation that the tube for recovering the nucleic acid has a structure that can be detachably attached to the support plate.
[0016]
In the present invention, the biological sample refers to a sample containing nucleic acids such as blood, serum, buffy coat, urine, feces, active spinal fluid, semen, saliva, tissue, cells, and culture.
[0017]
The reagent used in the present invention is a reagent for extracting a nucleic acid, and includes a buffer containing a chaotropic substance, EDTA, Tris-HCl or the like. Examples of the chaotropic substance include a guanidine salt, sodium iodide, potassium iodide, sodium (iso) thiocyanate, and urea. They may be used in combination. The concentration is preferably about 1 to 10 mol / l. Guanidine thiocyanate, Triton X-100, Tris / HCl buffer are particularly preferred.
[0018]
Further, the reaction tank portion in the present invention is a portion for extracting nucleic acid from the biological sample using the reagent, and the drain cup portion is a portion for collecting a liquid containing unnecessary components at the time of performing the extraction. In some cases, the tube for collecting nucleic acid refers to a portion for collecting an extract containing the target nucleic acid. Further, the support plate refers to a plate-like plate for holding the reaction tank portion, the drain cup portion, and a tube for recovering the nucleic acid.
[0019]
Also, preferably, a discharge pipe for discharging the liquid in the reaction tank portion, a rotating member connected to the discharge pipe, and a discharge passage formed in the rotating member so that the liquid can be discharged by being inserted into the upper portion of the reaction tank. The liquid discharge passage is controlled by the rotating member.
[0020]
More preferably, it has a structure in which a pipe penetrating through the rotating member is formed integrally, and the liquid in the reaction tank is discharged by pressure applied from above the penetrating pipe of the rotating member.
[0021]
More preferably, the rotation member is controlled to rotate by a driving means provided outside, so that the liquid in the reaction tank can be selectively discharged to a drain cup portion or a collection tube.
[0022]
According to the above-mentioned means of the reaction tank and the rotating member, in the operation of injecting and discharging the liquid performed in the nucleic acid extraction process, when injecting the liquid through the through pipe of the rotating member, the injection nozzle or the tip of the injection tip is moved to the reaction tank. It can be carried out without being inserted into the inside, and the liquid in the reaction vessel is drain-cup or recovery-tube along the discharge channel formed by the discharge pipe and the rotating member by the pressure applied from the through pipe. Can be discharged to
[0023]
Here, as the material of the above-described reaction tank portion, the drain cup portion, and the tube for recovering nucleic acid constituting the container for nucleic acid extraction, conventionally used materials such as glass, polypropylene, polyvinyl chloride or A general resin coated with silicon is exemplified. Among them, polypropylene is particularly preferable because it has no nucleic acid adsorption. As for the shape, the reaction tank portion is preferably conical, the drain cup portion is cylindrical, and the tube for recovering nucleic acid is preferably a commonly used microtube.
[0024]
Hereinafter, the container of the present invention will be described with reference to the drawings.
[0025]
FIG. 2 is a diagrammatic sectional view showing one embodiment of the container for nucleic acid extraction of the present invention. The container for nucleic acid extraction includes a reaction tank portion 1 for mixing and reacting a biological sample and a reagent, a drain cup portion 2 for storing an unnecessary component liquid, a tube 3 for collecting nucleic acid, and discharging nucleic acid to a tube 3 for collecting nucleic acid. A lid 8 that forms a flow path to be rotated, a rotating member 6 that is formed by being inserted into the upper part of the reaction tank portion 1, a cap 9 that suppresses vertical play during rotation of the rotating member 6, and assembling and fixing the entire extraction container And a support plate 4 serving as a support plate. Reference numeral 7 denotes a drain cup / lid, and reference numeral 10 denotes a through pipe.
[0026]
FIG. 3 shows a detailed assembly and disassembly of one embodiment of the container of the present invention. In the figure, 1 to 10 are as described above. Reference numerals 11 and 12 denote arms, and reference numeral 16 denotes a hole. FIG. 4 is a view showing a support plate of the container for nucleic acid extraction of the present invention. In the figure, 13 is a hole, and 14 and 15 are notches. Using these, the structure will be described in more detail.
[0027]
The container for nucleic acid extraction is assembled based on the support plate 4 shown in FIG. First, a U-shaped notch 14 is provided on one side of the support plate 4, the drain cup portion 2 is inserted and fitted into the U-shaped notch, and an arm 11 integrally formed with the reaction tank portion 1 is provided. The drain cup portion 2 is fixed to the support plate 4 such that the hole 16 provided in the drain cup portion is inserted and coupled. Further, a lid 7 having a small hole for venting air is inserted and fitted into the upper open portion of the drain cup portion 2.
[0028]
On the other hand, an arm 12 is similarly formed on the opposite side of the arm 11 provided with the reaction tank, and a lid 8 that forms a discharge flow path to the nucleic acid recovery tube 3 is inserted into and engaged with the arm 12 and fixed. The nucleic acid collection tube 3 is inserted and fixed into the U-shaped notch 15 of the support plate 4.
[0029]
Next, the rotating member 6 connected to the discharge pipe 5 is inserted and fitted into the open upper part of the reaction tank portion 1. The rotating member 6 is integrally formed with a through pipe 10 and is used as a liquid injection passage to the reaction tank portion 1 or a passage for applying pressure from outside. Furthermore, a cap 9 provided with a screw on the inside is fixed to the reaction tank portion 1 by screw tightening in order to eliminate vertical play during rotation of the rotating member 6.
[0030]
FIG. 5 shows the rotating member 6, FIG. 5 (a) is a top view, and FIG. 5 (b) is a side view. The discharge pipe 5 is connected by being inserted and connected to the passage 17 of the rotating member to form a discharge flow path. Further, the inner diameter of the through pipe 10 provided in the rotating member has a tapered structure that becomes smaller toward the inside of the reaction tank portion.
[0031]
As described above, the nucleic acid of the present invention has a configuration in which a reaction tank portion for mixing and reacting a biological sample and a reagent, a drain cup portion for storing an unnecessary component liquid, and a tube for recovering nucleic acid are fixed to the same support plate. An extraction container is to be comprised. The collection tube 3 has a detachable structure because it needs to be removed when nucleic acid extraction is completed.
[0032]
Next, an example of a method for using the above-described container for nucleic acid extraction will be described. Here, the nucleic acid extraction method is based on the method using a magnetic carrier already described in JP-A-9-19292. That is, a biological sample containing a magnetic silica particle containing a superparamagnetic metal oxide, a nucleic acid, and a nucleic acid extraction solution are poured into the container of the present invention and mixed. The magnetic silica particles to which the nucleic acid is bound are separated from the liquid using a magnetic field (B / F separation), and the nucleic acid is finally eluted from the magnetic silica particle to which the nucleic acid is bound.
[0033]
FIG. 6 shows a schematic view of the use of the nucleic acid extraction container of the present invention when performing the above-described nucleic acid extraction method. A more detailed description will be made based on this. First, FIG. 6A shows a liquid injection step. That is, a dispensing nozzle or a filter tip 18 connected to a dispensing means (not shown) such as a syringe or a pipettor is positioned at the entrance of the penetrating pipe 10, and the liquid is injected. In this case, since the tip of the dispensing nozzle or the tip of the filter tip is not inserted to the inside of the reaction tank, there is no danger that factors that inhibit the reaction system will be mixed into the inside of the reaction tank from the outside.
[0034]
In this step, specifically, a nucleic acid-binding magnetic carrier such as magnetic silica particles, for example, a biological sample containing nucleic acid such as blood, and a nucleic acid extraction solution are injected.
[0035]
Next, FIG. 6B shows a mixing step. In this step, the magnetic silica particles, the biological sample containing nucleic acid and the nucleic acid extraction solution are placed inside the reaction vessel by the up and down movement of an external permanent magnet, in particular, here, the ring-shaped magnet 19 to move the magnetic carrier inside the reaction vessel. The whole liquid is mixed by stirring. Adjustment of the strength of the permanent magnet or the stirring time ensures that the entire liquid is sufficiently mixed.
[0036]
FIG. 6C shows a step of separating the magnetic carrier to which the nucleic acid is bound from the liquid using a magnetic field (B / F separation). This step can be performed using a permanent magnet. Specifically, a permanent magnet is brought close to the bottom of the reaction tank containing a liquid consisting of a biological sample, a nucleic acid extraction solution, and a magnetic carrier, and only the magnetic carrier is collected at the bottom of the reaction tank, and unnecessary component liquids such as a nucleic acid extraction solution Is to be separated.
[0037]
FIG. 6D shows a step of discharging the unnecessary component liquid. A pressure nozzle tip 22 connected to a pressure pump (not shown) is positioned at the entrance of the through pipe of the rotating member. When pressure is applied from the pressure pump in this state, the liquid inside the reaction tank is pushed out along the discharge flow path formed by the discharge pipe and the rotating member, and is discharged to the drain cup portion. At the same time, an exhaust nozzle 23 is installed in a small hole at the top of the drain cup, guides gas from the reaction tank, and collects and processes the waste gas in one place (not shown) such as a waste bottle. By doing so, the risk of contamination with other containers can be prevented. Specifically, only the solution such as the solution for nucleic acid extraction which has become unnecessary moves to the drain cup portion through the discharge channel while the magnetic carrier is collected by the magnet at the bottom of the reaction tank.
[0038]
Next, the magnetic carrier is washed several times with ethanol. This can be performed by executing the above-described steps of injection, mixing, B / F separation, and discharge exactly in the same manner.
[0039]
In the step of drying the magnetic carrier, the unnecessary component liquid is discharged, and then the hot air can be passed through the inside of the reaction tank, or the reaction tank can be easily dried by heating from outside.
[0040]
Finally, the nucleic acid is eluted from the magnetic carrier, and the recovering step is performed by performing the above-mentioned washing and drying treatments, and then adding a low ionic concentration solution such as sterilized water to the magnetic carrier, thereby binding the nucleic acid to the magnetic carrier. Nucleic acids can be recovered from low ionic strength solutions. FIG. 6E shows a schematic diagram of this recovery step.
[0041]
In the step of recovering the nucleic acid, when the rotating member is rotated by 180 degrees by the external driving means, the discharge flow path formed by the discharge pipe and the passage of the rotating member is connected from the drain cup portion to the collection tube side.
[0042]
Therefore, the nucleic acid extraction container has a structure in which the liquid in the reaction tank can be selectively discharged to the drain cup portion or the collection tube by controlling the rotation of the rotating member.
[0043]
【The invention's effect】
The present invention is characterized in that a reaction tank part for mixing and reacting a biological sample and a reagent, a drain cup part for storing an unnecessary component liquid, and a tube for recovering nucleic acid are fixed to the same support plate, and the present invention The use of a container for nucleic acid extraction not only significantly prevents contamination than microtubes and the like used in conventional open systems, and also eliminates the risk of contamination from outside with risk factors that hinder the extraction reaction. Can be much less. Therefore, nucleic acids used for genetic diagnosis and the like can be extracted with high reliability.
[Brief description of the drawings]
FIG. 1 is a diagram showing common steps of a general nucleic acid extraction process.
FIG. 2 is a view showing a container for nucleic acid extraction of the present invention.
FIG. 3 is an exploded view of the nucleic acid extraction container of the present invention.
FIG. 4 is a view showing a support plate of the container for nucleic acid extraction of the present invention.
FIG. 5 is a view showing a rotating member of the container for nucleic acid extraction of the present invention ((a) top view, (b) side view).
FIG. 6 is a schematic view of a nucleic acid extraction method using the nucleic acid extraction container of the present invention ((a) injection step, (b) mixing step, (c) B / F separation step, (d). Unnecessary component liquid discharging step, (e) nucleic acid collecting step).
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Reaction tank part 2 Drain cup part 3 Collection tube 4 Support plate 5 Drain pipe 6 Rotating member 7 Drain cup lid 8 Lid 9 which forms a collection channel 9 Cap 10 Penetrating pipes 11 and 12 Arms 13 Holes 14 and 15 Notches 16 Hole 17 Passage 18 Dispensing nozzle or filter tip 19 Ring magnet 20 Magnet 21 Magnetic carrier 22 Pressurizing nozzle 23 Exhaust nozzle

Claims (3)

A reaction vessel part for mixing and reacting a biological sample and a reagent, a drain cup part for storing an unnecessary component solution, and a tube for recovering nucleic acid , which is a container for nucleic acid extraction constituted by fixing the same to a support plate , Furthermore, (1) a discharge pipe for discharging the liquid in the reaction tank portion and a discharge pipe formed by penetrating the discharge member and integrally forming the discharge pipe are inserted into the upper portion of the reaction tank, and from the upper portion of the penetration pipe of the rotation member, A structure in which the liquid in the reaction tank is discharged by the applied pressure. (2) The rotation of the rotating member is controlled by a driving means provided outside, and the liquid in the reaction tank is selectively discharged to a drain cup portion or a collection tube. And a structure capable of extracting nucleic acids. 2. The container for nucleic acid extraction according to claim 1, wherein the tube for recovering nucleic acid has a structure that can be detachably attached to a support plate. 3. The container for nucleic acid extraction according to claim 1 , wherein a driving means is provided independently and outside of the container.

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