WO2002042430A1

WO2002042430A1 - Nucleic acid extraction device

Info

Publication number: WO2002042430A1
Application number: PCT/JP2000/008338
Authority: WO
Inventors: Naruo Watanabe; Yoshihiro Nagaoka; Yukiko Ikeda; Teruhisa Akashi; Yuji Miyahara
Original assignee: Hitachi, Ltd.
Priority date: 2000-11-27
Filing date: 2000-11-27
Publication date: 2002-05-30
Also published as: JPWO2002042430A1

Abstract

A nucleic acid extraction device so constituted as to extract nucleic acid by moving a nucleic acid-containing solution, by providing a plurality of tanks on a rotating disk, connecting each tank to each other with a flow path provided with an open/close mechanism, and sequentially separate and extract specified components from solutions in respective tanks. An automatic treating by means of a plurality of tanks connected with open/close mechanism-carrying flow paths can treat an aqueous solution containing a large amount of nucleic acid with no foreign matters mixed in to ensure high-sensivity, high-accuracy extraction of nucleic acid and reduce nucleic acid extracting cost and time.

Description

Specification

Nucleic acid extraction equipment. Technical field

The present invention relates to a nucleic acid extraction device for extracting nucleic acids from cells, microorganisms, viruses, and the like in an aqueous solution.

Background art

There is a genetic diagnosis that uses a nucleic acid to diagnose a subject from this pattern. In this genetic diagnosis, nucleic acids are extracted from body fluids, tissues, and the like collected from a subject, and diagnosed by pattern analysis of the nucleic acids. For this reason, nucleic acid extraction must be performed accurately. Examples of conventional techniques relating to nucleic acid extraction include, for example, “Bio-experimental illustrations: Basics of molecular biology experiments (1995, Shujunsha) P113-P122” The prior art 1) discloses a method in which an aqueous solution containing a nucleic acid is held in a small container called a tube, and this is manually injected with various treatment liquids, stirred, and repeatedly centrifuged. As a device that automates this operation, it is marketed as Toyobo Co., Ltd. (Nucleic acid automatic separation device, NS-100).

In addition, as a conventional technique 2, “Micro 'Total Analysis System' 2000 Proceedings (2000), P239-P224” is a solution holding tank and a reaction tank on a resin circular substrate. A method is disclosed in which a microchannel and a heating element are provided, and a centrifugal force generated when a circular substrate is rotated is used to cause a reaction and a liquid transfer to extract nucleic acid from whole blood and analyze a pattern of the nucleic acid.

In the above-mentioned conventional technology, the following problems are likely to occur, and it is technically difficult to avoid them. In other words, the method of holding and treating the aqueous solution containing nucleic acids and various solutions of the prior art 1 in a small container called a tube can increase the sample volume and perform simple processing. Raw There is a problem that it is difficult to extract accurately. Although a complicated mechanism such as a robot system is adopted to automate these operations, it is difficult to increase the equipment cost and improve the throughput of the processing. The method of processing on a resin-made circular substrate of the prior art 2 is that the aqueous solution containing nucleic acids and various solutions are all held on one substrate, and high-precision extraction is possible without contamination by foreign substances. There is a problem that the sample volume cannot be increased because the holding tank and the reaction microchannel are fine, and it is difficult to extract nucleic acids contained in the sample when the amount is small. Since it is difficult to mix two liquids that separate into an oil layer and an aqueous phase in a stationary state when mixing a sample or a solution, etc. Could not go to.

An object of the present invention is to realize an accurate and highly sensitive nucleic acid extraction device that does not contaminate foreign substances in an apparatus for extracting nucleic acid from an aqueous solution containing nucleic acid, and to provide it at low cost. Disclosure of the invention

The present invention provides a rotating disk with an aqueous solution containing nucleic acid, such as blood, provided with a plurality of tanks separated by a flow path or a partition, and connecting the respective tanks by operating an opening / closing mechanism provided in the flow path or the partition. Then, by rotating the disk and operating the opening / closing mechanism, the nucleic acid was extracted while being sequentially moved for each component.

In addition, all or a part of the flow path or the partition, the opening / closing mechanism, and the tank are made of a plastic material from which a part of the component is not eluted with respect to the chemical having a protein denaturing action. Furthermore, in order to separate the protein into any of the plurality of tanks, a predetermined amount of a drug having a denaturing effect on the protein or a drug which is more easily bound to water molecules than nucleic acid is previously enclosed. Here, the drug having a protein denaturing action is a solution obtained by mixing guanidine thiosinate, buenol, phenol, black form and isoamyl alcohol in a ratio of 25: 24: 1, respectively. Alternatively, it refers to chemicals that have the effect of breaking down a membrane made of a protein containing nucleic acids and extracting nucleic acids, such as a solution of chloroform and isoamyl alcohol mixed in a 24: 1 ratio.

In addition, a chemical that is easier to bind to a water molecule than a nucleic acid refers to a chemical such as ethanol or isopropyl alcohol that has an action of coagulating a nucleic acid dissolved in water when mixed with an aqueous solution containing a nucleic acid.

With the above configuration, the aqueous solution containing nucleic acids is stored in the tank for each component, and therefore, the aqueous solution containing a large amount of nucleic acids is not limited to the volume per unit length of the flow path connecting them. Can be processed. 'In addition, since all processes can be automated, accurate extraction can be performed without contamination. In addition, the tank holding the aqueous solution is separated by a partition wall, and this tank is manufactured by plastic injection shaping, so that the cost can be reduced. And since it can be used up in one extraction, there is no measurement error due to the difference of the measurement target. In addition, by separating the tank holding the aqueous solution with a partition and making this tank detachable, it is possible to set optimal conditions for the treatment in each tank, and it becomes possible to perform efficient extraction. Further, since the aqueous solution containing the nucleic acid is moved, the transport capacity is improved, and the throughput of extraction can be improved. Furthermore, if blood is treated as an aqueous solution containing nucleic acids using this centrifugal force, serum can be obtained by the effect of centrifugation, and thus the throughput of extraction can be improved. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that nucleic acid is dissolved from an aqueous solution containing nucleic acid according to the first embodiment of the present invention. FIG. 2 is an external view of a device for extracting nucleic acids from an aqueous solution containing nucleic acids according to the first embodiment, FIG. 2 is an external view for explaining a mechanism for centrifuging or stirring the device, and FIG. FIG. 4 is a view showing a procedure for extracting a nucleic acid from an aqueous solution containing a nucleic acid using the device according to the first embodiment, and FIG. 4 is a diagram showing a device for extracting a nucleic acid from an aqueous solution containing a nucleic acid according to the second embodiment. Fig. 5 is a diagram showing the relationship between the operation of a mechanism for centrifuging or agitating the device and the operation of an opening / closing mechanism in Fig. 5; FIG. 6 is a diagram illustrating the function of a device for extracting nucleic acid from an aqueous solution containing nucleic acids according to the first embodiment. FIG. 7 is a diagram illustrating the function of a device containing nucleic acids according to a second embodiment of the present invention. Debye to extract nucleic acid FIG. 8 is a diagram showing a procedure for extracting a nucleic acid using a device for extracting a nucleic acid from an aqueous solution containing a nucleic acid according to the second embodiment, and FIG. 9 is a diagram showing a procedure for extracting the nucleic acid according to the second embodiment. FIG. 4 is a diagram showing the relationship between the operation of a mechanism for centrifuging or agitating the device and the operation of an opening / closing mechanism in a device for extracting nucleic acid from a contained aqueous solution. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

FIG. 1 (a) shows a plurality of tanks formed on a rotating disk and connected by a flow path having an opening / closing mechanism for extracting nucleic acids from an aqueous solution containing nucleic acids. In this example, human whole blood is particularly described as an example of an aqueous solution containing nucleic acids. However, the same applies to biological fluids containing nucleic acids or aqueous solutions in which tissues containing nucleic acids are suspended in water. Can be extracted.

FIG. 1 (b) is an enlarged view of a portion of the tank and the flow channel in FIG. 1 (a). The disk 100 has a plurality of tanks formed therein. The disk 100 is provided with a mounting hole 102 for being mounted on a drive mechanism and rotated. Disk 1 0 0 has the following layers. A sample holding tank 104 for holding whole blood as a sample for analysis, a separation tank 106 for separating serum from whole blood, and an extraction for removing nucleic acids from a nuclear wall containing nucleic acids A tank 108, a collection tank 110 for collecting nucleic acids diffused in the aqueous solution, and a nucleic acid holding tank 112 for holding extracted nucleic acids. The sample holding tank 104 and the separation tank 106 are connected by a flow path 114. The separation tank 106 and the extraction tank 108 are connected by a flow path 116. The flow path 1 16 is provided with an opening / closing mechanism 122 for controlling the movement of the solution on the way. The flow path 118 connects the extraction tank 108 and the recovery tank 110, and includes an opening / closing mechanism 124 for controlling the movement of the solution in the middle thereof. The flow path 120 connects the collection tank 110 and the nucleic acid holding tank 112 and has an opening / closing mechanism 126 for controlling the movement of the solution in the middle thereof. Wiring 1 2 8, wiring 1 3 0, and wiring 1 3 2 are connected to the disc 1 0 0 through the hole 10 2 to control the respective opening and closing mechanisms 1 2 2, 1 2 4 and 1 2 6. Connected to an external (not shown) controller. Although the opening / closing mechanism of this embodiment is configured by an electromagnetic valve that opens and closes with an electromagnetic force, the present invention is not limited to this method, and a system that switches gears and the like with a single drive system to open and close may be used.

The sample holding tank 104 is provided with a hole 103 for injecting whole blood. Each tank except for this hole and each flow path are sealed to shut off outside air, preventing foreign matter from entering.

Fig. 2 shows a mechanism for generating centrifugal force by rotating the disk 10 ° including the tank and a mechanism for stirring the solution contained in the tank in the disk 100 °. . The disk 100, the rotating shaft 200 and the motor 202 are mounted on the disk 204. The disk 204 is rotationally driven by a rotating shaft 206 of the motor 208. In this embodiment, the center of the rotation axis 200 of the disk 100 and the center of the rotation axis 206 of the disk 204 are attached with a predetermined distance therebetween. In this way, it is necessary to provide a rotating shaft at intervals. Thus, as described later, the centrifugal force applied to each tank can be increased. That is, while rotating around the rotating shaft holding the disk 100, the whole of them is driven in a so-called planetary motion in which the center of the rotating shaft is shifted from the rotating shaft 206 of the disk 204. ,

The disk 204 is provided with wires 2 1 2, 2 1 4 and 2 16, which are connected to the wires 1 2 8 1 3 0 and 1 3 2 provided on the rotating disk 100. ing. In addition, wirings 218 and 220 for driving and controlling the motor 202 are provided on the disk 204. The operation of each opening / closing mechanism on the disk 100, the motor, and the motor 208 for rotating the disk 204 is centrally controlled by the control device 230 via the control line 232. .

FIGS. 3 (a) to 3 (f) are diagrams for explaining the operation of extracting nucleic acids from whole blood. In FIGS. 3 (a) to 3 (f), there is a protein denaturing effect for removing nucleic acids from the nuclear wall encapsulating nucleic acids, which is not shown in FIG. 1 or FIG. A predetermined amount of the drug 300 is held in the extraction tank 108, and the drug 302, which is easier to bind to water molecules than the nucleic acid, is stored in the recovery tank 110. The arrow 100 0 indicates the direction of the centrifugal force generated by the rotation of the disk 100.

FIG. 3 (a) shows a state in which the disk 100 is stationary. At this time, 310 whole blood collected is injected into the sample holding tank 104 from the injection hole 103.

Next, FIG. 3 (b) shows the case where the disk 100 is rotated near the separation tank 106 so that the centrifugal force becomes 150 G or more. The whole blood 310 moves from the sample holding tank 104 to the separation tank 106. When this rotation is continued for about 5 minutes, substances 3 14 other than serum are distributed on the outer periphery of the disc 100 and serum 3 12 is distributed on the rotation center side in the separation tank 106 by centrifugal force. At this time, the higher the rotation speed of the disk 100, the more the separation tank 106 The time for separating serum and components other than serum can be shortened. In order to send the separated high-purity serum to the next extraction layer 108 because of the separation state described above, the outlet of the flow path 114 in the separation tank should be It is desirable that the entrance of the flow path 1 16 be located on the side of the center of rotation of the disk 100. The separated serum 312 passes through the flow path 1 16, passes through the opening / closing mechanism 1 2 2 previously opened, and moves to the extraction tank 108.

FIG. 3 (c) shows a state in which nucleic acids are extracted from the serum 312 in the extraction tank 108. A predetermined amount of a drug 300 having a protein denaturing action is held in the extraction tank 108 in advance, and the drug 300 reacts with the serum 112. For example, when phenol is used as the drug 300 having a protein denaturing effect, when serum is sent, phenol 300 is initially converted to an oil layer in the extraction tank 108, and serum 12 is in a separated state as an aqueous layer. The amount of phenol 300 is preferably equal to or greater than the amount of serum 112 to be reacted.

As described above, since serum 310 and drug 300 are in a separated state, in addition to rotating disk 100 in motor 202, disk 204 is also in liquid. It was decided to rotate and stir at 08. At this time, the respective opening / closing mechanisms 122 and 124 in the middle of the flow path 116 and the flow path 118 are closed. The centrifugal force generated by the rotation of both disks is synthesized in the extraction tank 108 on the disk 100. For this reason, the rotation speed of the disk 100 and the disk 204 needs to be a value such that the direction of the centrifugal force does not depend on the rotation angle of the disk 100. For example, the extraction tank 108 is provided at a position 45 mm away from the center of the rotation axis 200 (the diameter of the extraction tank 108 is 6 mm), and the rotation axis 200 is the rotation axis. It is located at a distance of 51 mm from 206. When the rotation speed of the disk 100 is set to 500 rpm, the rotation speed of the disk 204 may be set to be higher than 200 rpm rp'm. The shape of the extraction tank 108 is shown in Fig. 1. In addition to the circular shape shown in FIG. 3, if the shape is a polygon such as a square, the effect of extracting nucleic acids is enhanced.

Next, FIG. 3 (d) shows that after the operation of extracting nucleic acids from serum 312 shown in FIG. 3 (c), the rotation of the disk 204 shown in FIG. This figure shows the state when 100 is rotated near the extraction tank 1108 so that the centrifugal force becomes 150 G or more. At this time, for example, when phenol is used as the drug 300 having a protein-degrading action, the phenol 300 has a small centrifugal force in the portion where the centrifugal force increases, and the aqueous solution 3 In between, albumin and globulin, which are proteins in serum (not shown) denatured by phenol 300, are distributed in layers. Next, when the opening / closing mechanism 124 in the flow path 118 is opened, the aqueous solution 316 containing nucleic acid moves from the extraction tank 108 to the recovery tank 110. Next, FIG. 3 (e) shows a so-called centrifugal separation operation of taking out a very small amount of the solution having an increased nucleic acid concentration from the aqueous solution 316 containing the nucleic acid. The recovery tank 110 previously holds a drug 302 that is easier to bind to water molecules than nucleic acids. Then, the chemical 300 and the aqueous solution 316 containing nucleic acid are reacted. For example, when ethanol alcohol is used as a drug 302 that is easier to bind to water molecules than nucleic acids, water molecules in the aqueous solution 3 16 and molecules of ethanol alcohol 302 bind to dissolve the nucleic acids. The amount of water that was being used is reduced. For this reason, nucleic acids are precipitated. Furthermore, since the density of the nucleic acid is higher than that of the surrounding water or ethanol alcohol 302, the centrifugal force 1 (precipitates in the direction of 300. The larger the centrifugal force 100, the faster the nucleic acid becomes. Is desirably 1000 g or more.The amount of ethanol alcohol 302 is 2.5 times larger than that of aqueous solution 3 16 to be reacted with ethanol. For this reason, the recovery tank 110 is designed so that its volume is larger than that of the extraction tank 108 and the separation tank 106. It is a deeper tank in size. Here, for example, when isopropyl alcohol is used as the chemical 302 that is easier to bind to a water molecule than a nucleic acid, this amount may be equal to the amount of the aqueous solution 316 to be reacted therewith.

According to the configuration shown in FIG. 1 to FIG. 3, the location where the centrifugal force 1000 becomes maximum is near the opening / closing mechanism 126 in the middle of the flow path 120.

Next, FIG. 3 (f) shows the operation of moving the nucleic acid precipitated near the opening / closing mechanism 126 in the middle of the channel 120 to the nucleic acid holding tank 112 together with the nearby aqueous solution. It is. Also at this time, it is desirable that centrifugal force of 1000 G or more is applied to the recovery tank 110. By opening and closing the opening and closing mechanism 126 in the middle of the flow path 120 for a very short time under this gravity, the solution having a high nucleic acid concentration can be moved to the nucleic acid holding tank 112. For example, if the centrifugal force is 100 000 G, the cross-sectional area of the flow path 120 is 1 mm2, and the opening and closing time is 1 ms, 50 microliters of solution will be contained in the nucleic acid holding tank 112. Can be maintained. In addition, when the volume of the nucleic acid holding tank 112 is set to a target volume in advance, it is not necessary to open and close the opening / closing mechanism 126 in a short time, and the solution is filled in the nucleic acid holding tank 112. The open / close mechanism 126 may be opened up to this point. FIG. 4 (a) shows the control sequence of each motor and the open / close mechanism. That is, the control device 230 drives the disk 210 to rotate the motor 210, the motor 204 rotates the disk 204, and the opening / closing mechanism 122 on the disk 100. 4 and 1 26 show the operation status in the processing state. However, the positional relationship of each tank on the disk 100 is as shown in Fig. 4 (b). The distance between the center of the rotation axis 200 and the center of the rotation axis 206 is 51 mm. Also, the horizontal axis of the graph is not real time, but the processing content shown at the bottom of the graph.

As described above, according to this example, the amount of nucleic acid contained in the solution in the nucleic acid holding tank 112 is proportional to the amount of whole blood injected into the sample holding tank 104. And increase. The amount of the solution in the nucleic acid holding tank 112 is limited to the amount of the solution localized near the opening / closing mechanism 126 in the middle of the channel 120. Therefore, when the amount of nucleic acid contained in the collected whole blood 310 is small, the amount of nucleic acid contained in the finally obtained solution 320 can be increased by increasing the amount of whole blood. Highly sensitive nucleic acid extraction can be performed.

Conversely, when the amount of nucleic acid contained in whole blood 310 is large, the amount of nucleic acid contained in final solution 320 can be adjusted by reducing the amount of whole blood. It can handle whole blood with a wide range of nucleic acid concentrations. In addition, since the whole blood, from 310 to solution containing a large amount of nucleic acid, is processed in a plurality of tanks and a flow path connecting them, accurate extraction is possible without the risk of foreign matter entering. it can.

In the embodiment described above, each tank, each flow path, and the inlet port 103 set on the circle 100 can be integrally formed by directly cutting out the material forming the disc 100. it can. If they are integrally formed, the disk 100 can be made smaller and the processing cost can be reduced. This means that the disc 100 can be easily made disposable, and the effect of eliminating contamination between samples can be obtained. Polypropylene or fluororesin is suitable as a material for forming the disk 100. In addition, any material that does not dissolve its components in whole blood and is not violated by a drug 300 having a protein denaturing effect can be applied. Further, when a polycarbonate resin having good formability is used as a material for forming the tank and the flow path, if this surface is coated with a fluororesin, the strength of the disk 100 and the intrusion of foreign matter during processing can be avoided. And the disk 100 can be formed at low cost. Further, as a material for forming the tank / flow path, a material that elutes trivalent ions such as aluminum can be used. By using the material, a three-dimensional shape such as a tank can be pressed. Production costs can be reduced.

In addition, each tank and each flow path provided on the disk 100 are formed separately by injection molding of polypropylene, and these are arranged on the disk 100 and connected by bonding. May be manufactured. When manufactured in this manner, the volume of each tank can be adjusted or selected according to the sample, the processing conditions in each tank can be optimized, and the nucleic acid can be extracted efficiently. can get.

FIG. 5 shows another embodiment. In this embodiment, a new flow path 1 16 0 and an opening / closing mechanism 1 2 are provided between the extraction tank 108 of the previous embodiment (referred to as the first extraction tank in this embodiment) and the collection tank 110. 20 and a second extraction tank 1 080 were installed. In the second extraction tank 1800, phenol is previously stored as a drug 30000 having a protein denaturing action. In the second extraction tank 1800, the aqueous solution containing the nucleic acid extracted in the first extraction tank 1108 is re-extracted with a chemical 30000. With this configuration, the amount of protein contained in the solution that moves to the recovery tank 110 can be further reduced as compared with the case of the previous configuration, and the effect of improving the accuracy of nucleic acid extraction can be obtained.

FIG. 6 shows the configuration of still another embodiment. In this configuration, between the collection tank 110 (referred to as the first collection tank in the present embodiment) and the nucleic acid holding tank 112, a new flow path 1200 and an opening / closing mechanism 124 are added. This is a configuration in which two tanks 110 are installed. In the second collection tank 110, ethanol alcohol 302 is held in advance similarly to the first collection tank 110, and the water containing nucleic acid is stored in the first collection tank 110. Take out a small amount of the nucleic acid with a higher nucleic acid concentration from the layer.After that, in the second recovery tank 110, mix this small amount of the solution with ethanol alcohol 302, perform centrifugation, and re- It was designed to take out a small amount of solution with a high concentration. Therefore, transfer to the nucleic acid holding tank 1 1 2 The amount of sugar contained in the moving solution can be further reduced, and the effect of improving the accuracy of nucleic acid extraction can be obtained.

In the above embodiment, an injection hole 103 for injecting whole blood as a sample and one processing equipment from the sample holding tank 104 to the nucleic acid holding tank 112 are provided on the disk 100. As described above, the same effect can be obtained by installing a plurality of the devices. Providing a plurality of processing facilities on a single disk has the effect of increasing the number of processes per unit time in the process of extracting nucleic acids from aqueous solutions containing a plurality of nucleic acids.

Hereinafter, another embodiment of the present invention will be described in detail with reference to FIG.

FIG. 7 shows a configuration in which a plurality of rooms are provided on a disk in a circumferential direction to extract nucleic acids from an aqueous solution containing nucleic acids, and a partition between the rooms is provided with an opening / closing mechanism. In this embodiment, the disk is described as rotating clockwise.

The disk 700 is provided with a plurality of tanks (rooms) partitioned by partition walls in the rotation direction and the circumferential direction. The disk 700 is provided with a hole 720 for mounting on a mechanism (rotary shaft) for rotating the disk 700. The disc also has a sample holding tank 706 for holding whole blood, a separation tank 712 for separating serum from whole blood, and a nucleic acid for removing nucleic acids from a nuclear wall containing nucleic acids. Extraction tank 7 18, collection tank 7 2 4 for collecting nucleic acids diffused in aqueous solution, and chemical tank 7 30 holding chemicals that bind water molecules more easily than nucleic acids And a nucleic acid holding tank 736 for holding the prepared nucleic acid. As shown in the figure, the separation tank 712 provided on the outer peripheral side of the sample holding tank 706 is fractionated by a partition wall 707, and both are controlled by a centrifugal force as an opening / closing mechanism and a spring 710. Are connected by a sealer 708. Separation tank 7 12 and extraction tank 7 18 are separated by partition wall 7 17, and both are connected by a centrifugal force as an opening and closing mechanism and a seal 7 14 controlled by a spring 7 16 . Extraction tank 7 1 8 and The recovery tank 724 is fractionated by a partition wall 723, and is connected by a sealer 702 and a sealer 721 controlled by a centrifugal force as an opening / closing mechanism and a spring 722. . Note that the partition wall 723 is also used as a partition wall of the sample holding tank 706 and the recovery tank. The collection tank 724 and the pharmaceutical tank 730 are separated by a partition 729, and both are connected by a centrifugal force as an opening / closing mechanism and a sealer 726 controlled by a panel 728. The recovery tank 724 and the nucleic acid holding tank 736 are connected by a seal 732 controlled by a centrifugal force as an opening / closing mechanism and a spring 734. An injection hole 704 for injecting whole blood is provided on the upper surface of the sample holding tank 706.

Next, the operation of nucleic acid extraction using the disk 700 will be described with reference to FIGS. 8 (a) to 8 (f).

In FIG. 8 (a), with the disk 700 still, the whole blood 750 is injected through the injection hole 704. At this time, the sealer 708 is closed, and the whole blood 750 is confined in the sample holding tank 706. In addition, in the extraction tank 718, a drug 755 having a protein denaturing action is held in advance. In the chemical tank 730, a drug 754 which is easier to bind to water molecules than nucleic acid is held. Next, the disk 700 is rotated (clockwise rotation). Then, as shown in FIG. 8 (b), when the centrifugal force at the outermost circumference of the disk 700 reaches a rotation speed of 150 G or more, the seals 7 08 and 7 1 4 is opened, and the whole blood 750 held in the sample holding tank 706 moves to the separation tank 712. In addition, the sealer 714 moves toward the outer peripheral side to the extent that whole blood does not flow into the extraction tank 718, and is in an open state. In the separation tank 712, components 756 other than erythrocytes are distributed where the centrifugal force is large outside the disk 700, and the serum 758 is distributed further inside. Then, as the whole blood 750 moves from the sample holding tank 706 to the separation tank 712, the serum 758 overflows from the open end of the sealer 714 to the extraction tank 718. To go. 'this At this time, if the opening interval of the sealer 714 is set so that components 756 other than red blood cells cannot exceed the sealer 714, only the serum moves to the extraction tank 718. become.

Next, in FIG. 8 (c), the serum 758 has been moved to the extraction tank 718, and the rotation speed of the disk 700 has been reduced. It shows a closed state. A predetermined amount of a drug 752 having a protein denaturing action is held in the extraction tank 718 in advance. When, for example, phenol is used as the drug 752 having a protein denaturing effect, in the extraction tank 718, the phenol 752 is separated as an oil layer and the serum 758 is separated as an aqueous layer. Here, the amount of phenol 752 is desirably equal to or greater than the amount of serum 758 to be reacted therewith.

Next, phenol 752 and serum 758 are stirred to extract nucleic acids in serum 758. Therefore, the disk 100 in Fig. 2 is replaced by the disk 700, and in addition to the state in which the disk 700 is rotated by the motor 202 around the rotation axis 200, these are mounted. The disc 204 thus formed was rotated by a motor 208 around a rotation axis 206 to stir using planetary motion. At this time, the rotation speed of the disk 700 and the disk 204 is the centrifugal force generated by the rotation of the disk 700 and the disk 204 at the outermost periphery of the disk 700 of the extraction tank 718. A value is used so that the direction of the synthesized centrifugal force does not depend on the rotation angle of the disk 700. For example, if the radius of the disk 700 is 50 mm, the distance between the rotation axis 200 and the rotation axis 206 is 53 mm, and the rotation speed of the disk 700 is 500 rpm The rotation speed of the disk 204 may be higher than 2000 rpm. However, the rotation speed is set so that the sealers 708 and 714 are closed.

After the stirring, the rotation of the disk 204 shown in FIG. 2 is stopped, and the rotation speed of the disk 700 is increased. This allows the extraction tank 7 1 8 The phenol 752 is distributed where the centrifugal force is large, and the aqueous solution 760 containing nucleic acids is distributed further inside, while the phenol 752 is in the serum denatured by the phenol 752. Albumin / globulin, which is a protein (not shown), is distributed in layers.

Next, FIG. 8 (d) shows an operation of transferring the aqueous solution 760 containing a large amount of nucleic acid distributed in the extraction tank 718 to the recovery tank 724. At this time, the disk 700 has a greater number of rotations than the phenol 752 which is a chemical having a protein attribute action in the extraction tank 718 and the aqueous solution 760 containing nucleic acid. In step 12, the rotation speed is higher than the number of rotations for separating serum 758 and components 756 other than red blood cells, and a centrifugal force of about 300 G is generated in the collection tank. At this time, of the sealer 720 and the sealer 721 connecting the extraction tank 7 18 and the recovery tank 7 2 4, the sealer 7 2 1 moves according to the centrifugal force, A gap is generated between the sealing element 720 and the aqueous solution 760 from the gap to the recovery tank 724. However, a gap is also formed between the sealer 708 and the sealers 7 and 14 at the same time, but components 756 other than erythrocytes and chemicals 752 that have a protein denaturing action It does not move from 18.

Next, FIG. 8 (e) shows an operation of taking out a very small amount of a solution having a high nucleic acid concentration from an aqueous solution 60 containing nucleic acids held in the recovery tank 724. At this time, the rotation speed of the disk 700 is set to be higher than the rotation speed of the disk 700 in FIG. 8 (d) so that a centrifugal force of approximately 400 G is generated. At this time, of the sealer 720 and the sealer 721 connecting the extraction tank 718 and the recovery tank 724, the sealer 721 falls down according to the centrifugal force. At the same time, the sealer 720 is lowered so as to close the gap with the sealer 721, and the recovery tank 724 is sealed while holding the aqueous solution 760. Furthermore, increasing the number of revolutions of the disk 700 so that centrifugal force of about 500 G is generated To At this time, the seal 7 2 6 connecting the collection tank 7 2 4 and the chemical tank 7 30 is lowered, and a chemical that binds more easily to water molecules than the nucleic acid held in the chemical tank 7 30 7 5 4 moves to the collection tank 7 2 4. Thereby, an aqueous solution 762 reacted with the aqueous solution 760 is obtained. In this case, for example, ethanol alcohol was used as the chemical 754 which is easier to bind to water molecules than nucleic acids. As a result, in the aqueous solution 762, the volume of water in which the nucleic acid is dissolved due to the combination of the water molecule and the ethanol alcohol molecule is reduced. As a result, the nucleic acid precipitates, and the density of the nucleic acid becomes larger than that of surrounding water or ethanol alcohol, and the nucleic acid precipitates in a direction larger than the centrifugal force. This precipitation takes less time as the centrifugal force increases. Further, it can be applied to the precipitation of an aqueous solution 760 having a low nucleic acid concentration. It is preferable that the centrifugal force be 1000 G or more. Also, the amount of ethanol alcohol 754 is desirably 2.5 times or more of the aqueous solution 760 to be reacted therewith. For this reason, the recovery tank 724 is deeper in size than the extraction tank 718 and the separation tank 712 so as to have a larger volume.

Next, Fig. 8 (f) shows that the nucleic acid precipitated by centrifugal force in the aqueous solution 762 held in the recovery tank 724 and the slight aqueous solution around it are transferred to the nucleic acid holding tank 736. FIG. With a centrifugal force of 1000 G or more, the sealer 732 lowers, and the nucleic acid precipitates and the solution having a higher concentration moves to the nucleic acid holding tank 736. If the centrifugal force is 1000 G or more, the sealer 732 remains lowered, and the nucleic acid continues to accumulate in the nucleic acid holding tank 7336 during centrifugation with this centrifugal force. Then, when the rotation of the disk 700 is reduced and stopped, the sealer 736 is closed when the diameter of the disk 700 is cut, so that the nucleic acid diffuses again from the nucleic acid holding tank 736. I will not go. Fig. 9 (a) shows the motor 210 that the control device 230 rotates the disk 700, the motor 208 that rotates the disk 204, and the control device 230 on the disk 700. It shows the operation of the seals 7 08, 7 14, 7 20, 7 21, 7 26, and 7 36. However, the positional relationship of the sealers 7 08, 7 14, 7 20, 7 21, 7 26, and 7 36 on the disk 700 is as shown in Fig. 9 (b). It is. Here, the distance between the rotation axis 200 and the rotation axis 206 is 53 mm, and the diameter of the disk 700 is 100 mm. The horizontal axis of the graph is not real time, but the processing contents shown at the bottom of the graph.

As described above, according to the present embodiment, since the tank formed on the disk 700 is partitioned by the partition walls, the amount of whole blood to be processed can be further increased, and the highly sensitive nucleic acid can be obtained. An extraction can be performed. Also, if the entire disc 700 including each tank is manufactured by injection molding of polypropylene or the like, the production cost of the disc 700 can be reduced. Further, if the entire disc 700 including each tank is extruded and shaped with polycarbonate resin, and the surface is coated with a fluororesin, the strength of the disc 700 and entry of foreign matter during processing can be avoided. Furthermore, the disk 700 can be constructed at low cost. This is effective even when the amount of whole blood to be processed is small.If the polycarbonate resin surface is embossed to form each tank and the surface is coated with fluororesin, a disk 700 can be constructed at lower cost. can do.

In addition, in the stirring process in which the disk 204 on which the entire disk is mounted is rotated by the motor 208 while the disk 700 illustrated in FIG. 2 is rotated by the motor 202, fluorine is contained in the extraction tank 718. If a stirrer such as a small piece of resin is inserted in advance, the effect of stirring can be enhanced and the stirring time can be shortened. In addition, if the motor 210 that rotates the disk 700 is intermittently rotated and stopped and stirred without rotating the disk 204 with the motor 208, the device configuration can be simplified. Equipment costs can be reduced.

The same effect can be obtained even if a plurality of processing facilities from the hole 704 and the sample holding tank 706 to the nucleic acid holding tank 736 are installed on the disk 700. Wear. By providing a plurality of processing facilities, the effect of increasing the number of processes per unit time in the process of extracting nucleic acids from an aqueous solution containing a plurality of nucleic acids can be obtained.

Claims

The scope of the claims

1. A tank is provided on a disk, and the disk is rotated to extract nucleic acid from a solution using centrifugal force generated by rotation.

A nucleic acid extraction device, comprising: providing a plurality of the tanks; providing a channel having an opening / closing mechanism between the tanks; and sequentially moving the tank for each component in the solution to extract nucleic acids.

2. In a nucleic acid extraction apparatus for extracting a nucleic acid from a solution containing a nucleic acid, a sample holding layer for injecting the solution containing the nucleic acid on a disk and storing a predetermined amount thereof, and a separation tank for separating serum from the solution containing the nucleic acid An extraction tank for extracting nucleic acids from the separated serum, a collection tank for collecting the extracted nucleic acids, and a nucleic acid holding tank for holding the collected nucleic acids, and a flow path or partition between the tanks. An opening / closing mechanism provided in the flow path or the partition wall, and the disk is rotated to extract a nuclease using the action of centrifugal force.

3. All or a part of the flow path or partition, the opening / closing mechanism, and each tank are made of a plastic material that does not elute a part of its components with respect to a drug having a protein denaturing action. 3. The nucleic acid extraction device according to claim 2, wherein

4. The nucleic acid extraction device according to claim 2, wherein the opening and closing mechanism opens and closes depending on the magnitude of centrifugal force.

5. The nucleic acid extraction device according to claim 2, wherein the opening and closing mechanism opens and closes by an electromagnetic force.

6. The nucleic acid extraction device according to claim 1, wherein a force for moving the solution between the tanks is generated by centrifugal force.

7. The extraction tank is characterized in that a predetermined amount of a drug having a protein denaturing effect is held in advance in order to remove the nucleic acid from the nuclear wall containing the nucleic acid. The nucleic acid extraction device according to claim 2, wherein the nucleic acid extraction device is characterized in that:

8. The nucleic acid extraction device _{c9 according} to claim 2, wherein the recovery tank holds a chemical that is easier to bind to water molecules than nucleic acids. 9. The disk according to any one of claims 1 to 8, wherein the disc and a driving device for rotating and driving the disc are mounted on a disc separately provided with a driving device to increase the centrifugal force. Item 10. The nucleic acid extraction device according to Item 8.

10. The sample holding tank, the separation tank, the extraction tank, the recovery tank, and the nucleic acid holding tank are sequentially arranged on the outer peripheral side from the rotation center side of the rotating disk, and an opening / closing mechanism is provided between each tank. 3. The nucleic acid extraction device according to claim 2, wherein the nucleic acid extraction device is connected by a channel.