Cartridge, nucleic acid extraction, purification and separation system, and nucleic acid extraction method

Abstract

To provide cartridges, nucleic acid extraction, purification and separation systems, and nucleic acid extraction method capable of extracting nucleic acid.SOLUTION: Provided is a cartridge 100 for extracting nucleic acid using magnetic beads 80, configured with a first tube portion 10 provided with a first flow path 11 having a first inner diameter D1, and a second tube portion 20 provided with a second flow path 21 communicating with the first tube portion 10 via a connecting portion 40, having a second inner diameter D2 larger than the first inner diameter D1, and communicating with the first flow path 11, it being possible to take out the magnetic beads 80 from a liquid 50 introduced into the cartridge 100 onto the first tube portion 10 side, by the magnetic force acting from the outside, with the liquid 50 held on the connection portion 40 side of the second tube portion 20, and the amount of the liquid 50 held on the connection portion 40 side of the second tube portion 20 is greater than the amount of the liquid 50 contained in the magnetic beads 80 taken out onto the first tube portion 10 side.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cartridge, a nucleic acid extraction, purification and separation system, and a nucleic acid extraction method. [Background technology]

[0002] Patent Document 1 discloses a method for washing nucleic acid-binding solid phase carriers with adsorbed nucleic acids in a cartridge by moving them by changing the direction of application of an external magnetic field outside the cartridge. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2017-176023 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in the method described in Patent Document 1, nucleic acid-binding solid phase carriers are dispersed in a liquid reagent, specifically, a washing solution, and then a magnet is used to move the nucleic acid-binding solid phase carriers along a cartridge filled with the washing solution and a separation means, resulting in the washing solution being carried over to the next step. Furthermore, when amorphous metal is used for the magnetic beads that make up the nucleic acid-binding solid phase carriers, needle-like structures form along the magnetic field lines, resulting in a large amount of washing solution being carried over to the next step. As a result, the nucleic acid concentration decreases, reducing detection sensitivity and potentially inhibiting enzyme reactions or PCR. [Means for solving the problem]

[0005] The cartridge is a cartridge for extracting nucleic acids using magnetic beads, and is configured to include a first tubular section provided with a first flow path having a first inner diameter, and a second tubular section communicated with the first tubular section via a connection section and provided with a second flow path having a second inner diameter larger than the first inner diameter and communicating with the first flow path; the second flow path of the second pipe portion has a tapered portion that gradually becomes smaller toward the connecting portion,By using an external magnetic force, the magnetic beads can be removed from the liquid introduced into the cartridge to the first tube side, while the liquid is retained on the connection side of the second tube part.

[0006] A nucleic acid extraction, purification, and separation system includes a container having a first tubular section provided with a first flow path having a first inner diameter, and a second tubular section connected to the first tubular section via a connection section and provided with a second flow path having a second inner diameter larger than the first inner diameter and connected to the first flow path; a liquid containing magnetic beads introduced into the second flow path of the container; a magnet provided outside the container and causing the magnetic beads to generate a magnetic force; and a movement mechanism that changes the relative positional relationship between the magnet and the first and second tubular sections of the container in the extension direction of the first and second tubular sections of the container; the second flow path of the second pipe portion has a tapered portion that gradually becomes smaller toward the connecting portion, The moving mechanism changes the relative positional relationship between the magnet and the first and second tube portions of the container from a state in which the magnet corresponds to the second tube portion, to a state in which the magnet corresponds to the connection portion, and then to a state in which the magnet corresponds to the first tube portion, so that the magnetic beads bound to nucleic acids can be removed from the liquid to the first tube portion side while the liquid is retained on the connection portion side of the second tube portion.

[0007] The nucleic acid extraction method includes: a first tubular section provided with a first flow path having a first inner diameter; and a second tubular section connected to the first tubular section via a connecting section and having a second inner diameter larger than the first inner diameter. and has a tapered portion that gradually becomes smaller toward the connecting portion.and a second tube section having a second flow path communicating with the first flow path, the second tube section having a liquid containing magnetic beads introduced into the second flow path; a magnet arranged outside the first tube section and the second tube section is changed in relative position from a state in which the magnet corresponds to the second tube section in the extension direction of the first tube section and the second tube section to a state in which the magnet corresponds to the first tube section, and the magnetic beads in the second flow path are passed through the connecting section using the magnetic force acting between the magnetic beads and the magnet; and a step of extracting the magnetic beads from the liquid to the first tube section, with the liquid held on the connecting section side of the second tube section in an amount greater than the liquid contained in the magnetic beads extracted to the first tube section. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a cross-sectional view showing the configuration of a cartridge and a nucleic acid extraction, purification, and separation system. [Figure 2] 1 is a schematic diagram showing the structure of a nucleic acid-binding solid phase carrier. [Figure 3] 1 is a flowchart showing a nucleic acid extraction method. [Figure 4] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 5] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 6] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 7] 10 is a graph showing the relationship between the inner diameter of the first flow path and the amount of liquid carried over. [Figure 8] FIG. 1 is a cross-sectional view showing the configuration of a nucleic acid extraction, purification, and separation system. [Figure 9] 1 is a flowchart showing a nucleic acid extraction method using a cartridge. [Figure 10] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 11] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 12] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 13] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 14]1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 15] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 16] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 17] 1 is a cross-sectional view showing a nucleic acid extraction method. [Figure 18] FIG. 10 is a schematic diagram showing the configuration of a modified cartridge. DETAILED DESCRIPTION OF THE INVENTION

[0009] First, with reference to FIGS. 1 and 2, the configuration of the cartridge 100, the configuration of the nucleic acid 71 extraction, purification and separation system 1000, and the structure of the nucleic acid-binding solid phase carriers (hereinafter referred to as magnetic beads 80) will be described.

[0010] The cartridge 100 and nucleic acid 71 extraction, purification, and separation system 1000 are used as one method for purifying nucleic acid 71, such as a target substance or target component, from a liquid 50 in diagnosis and various tests in the medical and bio fields.

[0011] Specifically, for example, in a nucleic acid extraction process, which is a pre-process of a PCR test, or a cell extraction process, which is a pre-process of an antibody test, various biological materials are extracted and purified by magnetic separation (B / F separation) using minute magnetic beads 80. In order to efficiently extract nucleic acids 71, a method is used in the pre-process of the PCR method in which magnetic beads 80 capable of carrying nucleic acids are used to extract nucleic acids 71 ​​by applying magnetic force.

[0012] As shown in Figure 1, the cartridge 100 is used to adsorb nucleic acids 71 ​​onto magnetic beads 80 (see Figure 2), wash and purify the magnetic beads 80 with adsorbed nucleic acids 71, and extract the magnetic beads 80 with adsorbed nucleic acids 71, and is provided with a container 30 and a liquid 50 introduced into the container 30.

[0013] The container 30 is formed in a hollow cylindrical shape. Specifically, the container 30 includes a first pipe section 10 provided with a first flow path 11 having a first inner diameter D1, and a second pipe section 20 provided with a second flow path 21 having a second inner diameter D2 larger than the first inner diameter D1. The first pipe section 10 and the second pipe section 20 are in communication with each other via a connecting section 40. The container 30 is made of, for example, resin.

[0014] The first flow path 11 is formed with a first inner diameter D1 from the end 10a of the first pipe portion 10 to the connection portion 40. The diameter of the first inner diameter D1 is, for example, 1 mm to 2.5 mm.

[0015] The second flow path 21 is formed in a tapered shape that gradually decreases from the second inner diameter D2 from the end 20a of the second pipe section 20 to the connecting section 40. The tapered portion is referred to as the tapered portion. The angle θ of the second flow path 21 is, for example, 10° to 70°.

[0016] 1, the cartridge 100 includes a portion for accommodating the adsorption solution, a portion for accommodating the cleaning solution, a portion for accommodating the elution solution, and a separation layer portion for separating these portions so that they do not mix. Examples of the separation layer include air, silicone oil, a hydrophobic component, and a plastic partition wall.

[0017] The adsorption liquid is a liquid that serves as a site for adsorbing the nucleic acid 71 onto the magnetic beads 80. The adsorption liquid is an aqueous solution containing a chaotropic substance.

[0018] The washing liquid is a liquid for washing the magnetic beads 80 to which the nucleic acids 71 ​​are adsorbed. The washing liquid is, for example, a low salt concentration aqueous solution such as a buffer solution or an alcohol. In this embodiment, for example, ethanol is used.

[0019] The elution liquid is a liquid that separates the nucleic acid 71 from the magnetic beads 80 to which the nucleic acid 71 is adsorbed, and elutes the nucleic acid 71 into the elution liquid. For example, pure water is used as the elution liquid.

[0020] 1 contains magnetic beads 80. The liquid 50 includes a nucleic acid extraction solution, a washing solution, and the like.

[0021] 2, the magnetic bead 80 has a core-shell structure in which the surface of a magnetic core 81 is covered with a silicon oxide (SiO2) film 82. The magnetic core 81 and the silicon oxide film 82 are collectively referred to as the magnetic bead 80.

[0022] The magnetic beads 80 are a substance capable of adsorbing nucleic acids 71 ​​such as DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid), i.e., holding them by reversible physical and chemical bonding. When extracting nucleic acids 71, powdered magnetic beads 80 are used.

[0023] The magnetic core 81 is a particle having magnetic properties. The magnetic core 81 is an Fe-based amorphous (non-crystalline) magnetic metal particle. Therefore, the atomic arrangement of the magnetic core 81 is irregular, and the magnetic core 81 contains almost no crystalline structure or grain boundaries inside. The magnetic core 81 may also be an amorphous metal (amorphous alloy) containing Fe (iron), Cr (chromium), Si (silicon), and B (boron).

[0024] The magnetic cores 81 have a relatively high electrical resistance and a low coercive force due to the irregular atomic arrangement, and therefore do not easily aggregate with each other when no magnetic field is applied, and can be dispersed uniformly in the liquid 50.

[0025] The saturation magnetization of the magnetic core 81 made of Fe-based amorphous metal is, for example, 50 Am 2 / kg or more. Saturation magnetization is the value of magnetization that becomes saturated when the applied magnetic field is strong. By using such a magnetic core 81, it is possible to improve the ability to follow changes in the direction of magnetic force.

[0026] The size of the magnetic core 81 is, for example, an average particle diameter of 3 μm. The shape of the magnetic core 81 is spherical. The shape of the magnetic core 81 may have an elliptical or polygonal cross section.

[0027] The film thickness of the silicon oxide film 82 covering the surface of the magnetic core 81 is, for example, 5 nm to 100 nm. The silicon oxide film 82 coats the surface of the magnetic core 81. The silicon oxide film 82 specifically adsorbs the nucleic acid 71 in an aqueous solution containing a chaotropic substance.

[0028] The magnetic metal particles of the magnetic core 81 can be formed by, for example, an atomization method. Methods for forming the silicon oxide film 82 on the surface of the magnetic core 81 include, for example, a sol-gel method, a CVD (Chemical Vapor Deposition) method, a sputtering method, and a laser ablation method.

[0029] As shown in FIG. 1, the nucleic acid 71 extraction, purification, and separation system 1000 includes the above-mentioned cartridge 100 and a movement mechanism 300 that moves the magnet 200 along the extension direction of the container 30.

[0030] The magnet 200 is provided on the outside of the container 30. The magnet 200 is, for example, a permanent magnet. Alternatively, an electromagnet may be used. The magnet 200 applies a magnetic field to the magnetic beads 80 contained in the liquid 50 introduced into the cartridge 100. This allows the magnetic beads 80 to be freely manipulated by controlling the magnetic field from outside the container 30, without having to come into direct contact with them. Note that, because the magnetic beads 80 are made of a highly magnetized Fe-based amorphous metal, when a magnetic field is applied, they exhibit a needle-like structure along the magnetic field lines, as shown in FIG. 1.

[0031] By moving the magnet 200 along the moving mechanism 300, the magnetic beads 80 bound to the nucleic acids 71 ​​can be moved from the liquid 50, in other words, the magnetic beads 80, from the second tube section 20 to the first tube section 10. The magnetic field gradient of the magnetic force produced by the magnet 200 is, for example, 4 T / m to 237 T / m. The force acting on the magnetic beads 80 is determined from the saturation magnetization and weight of the magnetic core 81, and the applied magnetic field gradient.

[0032] The moving mechanism 300 is not limited to a configuration that moves the magnet 200 relative to the cartridge 100, in other words, the container 30, but may be configured to change the relative positional relationship between the magnet 200 and the container 30, for example, the magnet 200 may be fixed and the container 30 may be moved.

[0033] As described above, because the second flow path 21 of the cartridge 100 has a tapered shape, when the magnetic beads 80 are moved from the second tube section 20 toward the first tube section 10, friction occurs between the inner wall 21a of the second flow path 21 and the liquid 50. This slows down the movement speed of the liquid 50, and the liquid 50 and the magnetic beads 80 begin to separate. Furthermore, when the magnet 200 is moved toward the first tube section 10, only the magnetic beads 80 to which the nucleic acids 71 ​​are bound can be moved to the first tube section 10, while the liquid 50 remains in the connection section 40. This makes it possible to prevent the liquid 50 from being carried over to the next process.

[0034] Next, the nucleic acid extraction method will be described with reference to FIGS.

[0035] As shown in FIG. 3, in step S11, liquid 50 is introduced into cartridge 100. Specifically, as shown in FIG. 4, liquid 50 containing magnetic beads 80 is introduced from end 20a of second tube section 20. After the washing step, magnetic beads 80 made of Fe-based amorphous metal with strong magnetization are covered with a large amount of liquid 50. As described above, liquid 50 contains magnetic beads 80 including magnetic cores 81, a nucleic acid extraction solution, a washing solution, etc. The state shown in FIG. 4 is the state in which magnet 200 and second tube section 20 correspond to each other.

[0036] Next, in step S12, the liquid 50 containing the magnetic beads 80 is moved toward the first tube section 10. In other words, as shown in FIG. 5, the magnet 200 is moved by the moving mechanism 300, and the liquid 50 is passed through the connecting section 40. Specifically, a magnetic force acts between the magnetic beads 80 and the magnet 200, and the magnetic beads 80 move in response to the movement of the magnet 200. When the magnetic beads 80 move, the liquid 50 present between the magnetic beads 80 also moves. Note that the state shown in FIG. 5 is the state in which the magnet 200 and the connecting section 40 correspond to each other.

[0037] Next, in step S13, the magnetic beads 80 are separated and removed from the liquid 50. Specifically, as shown in Fig. 6, the magnet 200 is moved further toward the first tube section 10. Note that the state shown in Fig. 6 is the state in which the magnet 200 and the first tube section 10 correspond to each other.

[0038] Because the second flow path 21 has a tapered shape that narrows toward the connection portion 40, when the liquid 50 moves toward the connection portion 40, friction occurs between the inner wall 21a of the second flow path 21 and the liquid 50, slowing the movement of the liquid 50. Therefore, the liquid 50 and the magnetic beads 80 are separated, and the magnetic beads 80 bound to the nucleic acids 71 ​​can be moved to the next step while the liquid 50 is retained on the connection portion 40 side of the second tube portion 20. This makes it possible to prevent the liquid 50 from being carried over to the next step, and the nucleic acids 71 ​​can be detected. At this time, the amount of liquid 50 retained on the connection portion 40 side of the second tube portion 20 is greater than the amount of liquid 50 contained in the magnetic beads 80 extracted to the first tube portion 10 side.

[0039] For example, if the next step is an enzymatic reaction, the introduction of cleaning components may inhibit the enzymatic reaction. A typical example is the introduction of ethanol (a component of the cleaning solution) into PCR. According to this embodiment, the introduction of ethanol into the next step can be suppressed.

[0040] In this way, the magnetic beads 80 made of Fe-based amorphous metal can minimize the carryover of washing solution components into the eluate, allowing the volume of the eluate to be minimized, and as a result, the concentration of nucleic acids eluted in the eluate can be increased. As a result, the detection sensitivity of targets such as viruses and bacteria can be improved, and infections such as influenza and coronavirus, where the amount of nucleic acid is low, can be prevented from being overlooked.

[0041] Next, a preferred inner diameter of the first flow path 11 will be described with reference to FIG.

[0042] In the table shown in FIG. 7, the horizontal axis indicates the inner diameter of the first flow path 11, and the vertical axis indicates the amount of liquid 50 brought to the next process.

[0043] In the experimental process, first, 40 μL of pure water containing 40 wt% of magnetic beads 80 with a core-shell structure having a magnetic core 81 made of Fe-based amorphous metal and 100 μL of pure water were added to a polypropylene container and mixed.

[0044] Next, a neodymium magnet was applied to the outside of the container 30 to collect the magnetic beads 80. The magnet 200 was then moved along the container 30 until the magnetic beads 80 broke through the pure water interface. The magnetic beads 80 that had moved from the liquid into the air had a needle-like structure and were therefore surrounded by a large amount of pure water.

[0045] Next, the magnetic beads 80 containing the pure water were passed through a flow path with an inner diameter of 0.5 mm to 5.0 mm. The weight of the magnetic beads 80 containing the pure water after passing through was measured, and the weight of the magnetic beads 80, which was known in advance, was subtracted to calculate the volume of the pure water alone. The results are shown in the table in Figure 7.

[0046] These results show that when the inner diameter of the first flow path 11 is 1.0 mm to 2.5 mm, the amount of liquid carried over to the next process can be reduced to 8 μL to 43 μL. Note that when the inner diameter is 0.5 mm, the magnetic beads 80 clogged the first flow path 11 and could not be moved. It is believed that the strongly magnetized magnetic beads 80 exhibit a strong needle-like structure in a magnetic field, causing clogging within the first flow path 11. Note that when the inner diameter is 3 mm or greater, it was not possible to separate the pure water encased in the magnetic beads 80. However, by passing the magnetic beads 80 encased in liquid 50 through the first flow path 11 with a small inner diameter, the friction generated between the inner wall of the first flow path 11 and the liquid 50 separates the liquid from the magnetic beads. Therefore, the amount of liquid 50 carried over to the next process can be minimized.

[0047] That is, the inner diameter of the first flow path 11 is preferably within a range of 1 mm to 2.5 mm. According to this configuration, the first inner diameter D1 is formed within the above-described range, so that the magnetic beads 80 and the liquid 50 can be separated and the magnetic beads 80 can pass through the first flow path 11.

[0048] Next, the configuration of a nucleic acid 71 extraction, purification, and separation system 2000, which performs the entire process from introducing nucleic acid 71 to collecting eluate 112c, will be described with reference to Figure 8. The nucleic acid 71 extraction, purification, and separation system 2000 includes the configuration of the extraction, purification, and separation system 1000 described above. Below, the nucleic acid 71 extraction, purification, and separation system 2000, which performs the entire process from introducing nucleic acid 71 to collecting eluate 112c, will be described.

[0049] As shown in FIG. 8, the extraction, purification, and separation system 2000 includes a cartridge 110, a container pusher 120, a sample input means 130, and a magnet 200.

[0050] The cartridge 110 has an adsorption section 110a where a sample 70 containing nucleic acids 71 ​​is introduced and adsorbed to magnetic beads 80, a washing section 110b where the sample 70 containing magnetic beads 80 is washed, and an elution section 110c where the nucleic acids 71 ​​are eluted from the sample 70. The sample 70 is, for example, blood, throat swab, feces, culture medium, urine, etc.

[0051] In the cartridge 110, the second pipe section 20 is disposed on one side of the first pipe section 10, and the third pipe section 60 is disposed on the other side of the first pipe section 10. The third pipe section 60 has a third flow path 61 having a third inner diameter D3. The second flow path 21 has a structure in which its inner diameter is larger than that of the first flow path 11. Similarly, the third flow path 61 has a structure in which its inner diameter is larger than that of the first flow path 11.

[0052] The second flow path 21 and the third flow path 61 are filled with air, which serves as a liquid separating means when the flow paths are later filled with a liquid reagent 112. Silicon, polypropylene, polyethylene, etc. can be used for the inner walls of the first flow path 11, the second flow path 21, and the third flow path 61.

[0053] The third flow path 61 is formed in a tapered shape that gradually increases from the first inner diameter D1 (see FIG. 1) in the same manner as the above-described second flow path 21. The angle θ of the third flow path 61 is, for example, 10° to 70°.

[0054] The cartridge 110 also includes a first reagent storage section 111a for storing the liquid reagent 112 to be introduced into the adsorption section 110a, in this case, the adsorption liquid 112a, a second reagent storage section 111b for storing the liquid reagent 112 to be introduced into the washing section 110b, in this case, the washing liquid 112b, and a third reagent storage section 111c for storing the liquid reagent 112 to be introduced into the elution section 110c, in this case, the elution liquid 112c.

[0055] Blister packs (storage containers that release their contents when squeezed, like tablet medicine containers) are preferably used as these reagent storage units 111. By providing the reagent storage units 111, it is possible to prevent diffusion and contamination of liquid reagent components stored in the pretreatment cartridge.

[0056] The adsorption solution 112a is, for example, 6 M guanidine hydrochloride and 60% ethanol, the washing solution 112b is, for example, 70% ethanol, and the elution solution 112c is, for example, pure water.

[0057] The container pushing unit 120 has a first container pushing unit 120a for pushing the adsorption liquid 112a stored in the first reagent storage unit 111a into the adsorption unit 110a, a second container pushing unit 120b for pushing the cleaning liquid 112b stored in the second reagent storage unit 111b into the cleaning unit 110b, and a third container pushing unit 120c for pushing the elution liquid 112c stored in the third reagent storage unit 111c into the elution unit 110c. Note that the container pushing unit 120 is not particularly limited as long as it has the function of pushing the reagent storage unit 111.

[0058] The sample input means 130 is used to input the sample 70 or specimen into the cartridge 110, specifically into the adsorption section 110a. As the sample input means 130, for example, a pipette, a dropper, or the like is used.

[0059] As described above, the magnet 200 is used to apply a magnetic field to the magnetic beads 80 in the cartridge 110 and move the magnetic beads 80 in a desired direction. The moving mechanism 300 is not shown. The magnet 200 can be a neodymium magnet or an electromagnet.

[0060] Strongly magnetized amorphous metal magnetic beads are preferably used as the magnetic beads 80. The magnetic beads 80 may be present in a liquid, but in this embodiment, they are in a freeze-dried state.

[0061] 9 to 17, a nucleic acid extraction method using an extraction, purification, and separation system 2000 that performs processes from the introduction of nucleic acid 71 to the collection of eluate 112c will be described. The extraction, purification, and separation system 2000 includes the cartridge 100 described above.

[0062] As shown in Fig. 9, first, in step S21, a sample 70 containing nucleic acids 71 ​​is introduced into an adsorption solution 112a. Specifically, an extraction, purification, and separation system 2000 is prepared as shown in Fig. 10. Next, as shown in Fig. 11, the sample 70 is introduced into the adsorption section 110a of the cartridge 110 using the sample introduction means 130.

[0063] In step S22, nucleic acid 71 is adsorbed onto magnetic beads 80. Specifically, as shown in Fig. 12, first container pusher 120a is moved to push in first reagent storage section 111a of the blister pack in which adsorption liquid 112a is stored. This causes adsorption liquid 112a and sample 70 to mix.

[0064] Furthermore, as shown in Figure 13, a mixture 51 of adsorption liquid 112a and sample 70 comes into contact with magnetic beads 80, and by oscillating a pair of magnets 200, 200a, the magnetic beads 80 can be efficiently caused to collide with the target molecules in the sample 70.

[0065] In step S23, the magnetic beads 80 are washed. Specifically, as shown in Fig. 14, the second container pusher 120b is moved to push in the second reagent storage section 111b, which stores the cleaning solution 112b. Next, the magnet 200 is used to move the magnetic beads 80 to the cleaning section 110b. Thereafter, the magnet 200 is swung to disperse the magnetic beads 80 in the cleaning solution 112b.

[0066] In step S24, the magnetic beads 80 are moved from the second flow path 21 toward the first flow path 11. Specifically, as shown in FIG. 15, a magnetic field is applied to the magnetic beads 80 in the cleaning solution 112b by a magnet 200, causing the magnetic beads 80 to move toward the first flow path 11. This causes the magnetic beads 80 to pass through the first flow path 11. Most of the cleaning solution 112b is trapped in the second flow path 21 and is not carried into the first flow path 11 or beyond.

[0067] In step S25, the magnetic beads 80 are moved into the elution solution 112c, and the nucleic acids 71 ​​are eluted into the elution solution 112c. Specifically, as shown in FIG. 16, the third container pusher 120c is moved to push in the third reagent storage section 111c in which the elution solution 112c is stored. Next, the magnet 200 is used to move the magnetic beads 80 to the third flow path 61 having the elution section 110c. This brings the elution solution 112c into contact with the magnetic beads 80 and causes them to disperse. Thereafter, the magnet 200 is swung to efficiently elute the nucleic acids 71 ​​from the magnetic beads 80.

[0068] In step S26, the magnetic beads 80 are moved to the first flow path 11. In step S27, the magnetic beads 80 are subsequently moved to the second flow path 21. Specifically, as shown in FIG. 17, the magnet 200 is moved from the third flow path 61 toward the second flow path 21 to separate the magnetic beads 80 from the eluate 112c. At this time, the magnetic beads 80 follow the movement of the magnet 200 and pass through the first flow path 11, but the eluate 112c is trapped in the third flow path 61 and is not brought into the second flow path 21.

[0069] In step S28, the eluate 112c is collected. The eluate 112c is carried to the next step, such as PCR. When collecting the eluate 112c, the eluate 112c may be manually drawn out using a pipette, or the eluate 112c may be moved and removed by applying pressure to the adsorption section 110a (see FIG. 8), which is the sample input section. Furthermore, by providing a PCR reagent and a heating / cooling section near this flow path, the eluate 112c can be directly introduced into PCR without manual intervention.

[0070] It should be noted that the cartridge is not limited to the cartridge 110 shown in Fig. 8, and for example, a cartridge 120 shown in Fig. 18 may be used. Fig. 18 is a schematic diagram showing the configuration of a modified cartridge 120.

[0071] 18, cartridge 120 includes first tube 121 and second tube 122. A plug 123 is provided at the end of first tube 121. In cartridge 120, from the second tube 122 side, an adsorption liquid 124, a first oil 127a, a cleaning liquid 125, a second oil 127b, an elution liquid 126, and a third oil 127c are arranged in this order. Magnetic beads 128 are accommodated in second tube 122.

[0072] As described above, the cartridge 100 of this embodiment is a cartridge 100 that extracts nucleic acids 71 ​​using magnetic beads 80, and is configured to include a first tube section 10 having a first flow path 11 with a first inner diameter D1, and a second tube section 20 that is connected to the first tube section 10 via a connection section 40 and is provided with a second flow path 21 that has a second inner diameter D2 larger than the first inner diameter D1 and is connected to the first flow path 11.By using a magnetic force acting from the outside, the magnetic beads 80 can be extracted from the liquid 50 introduced into the cartridge 100 to the first tube section 10 side, with the liquid 50 retained on the connection section 40 side of the second tube section 20, and the amount of liquid 50 retained on the connection section 40 side of the second tube section 20 is greater than the liquid 50 contained in the magnetic beads 80 extracted to the first tube section 10 side.

[0073] According to this configuration, the liquid 50 containing the magnetic beads 80 introduced into (or contained within) the second flow path 21 is moved toward the first flow path 11 by passing through the connection part 40 using magnetic attraction, and the movement speed of the liquid 50 is slowed down by the action of friction generated between the inner wall 21a of the second flow path 21 and the liquid 50. Therefore, it is possible to efficiently extract and separate the magnetic beads 80 bound to the nucleic acids 71 ​​even when the amount of liquid 50 contained is small, and the magnetic beads 80 containing a small amount of liquid 50 can be transferred to the next step. This makes it possible to prevent the liquid 50 from being carried over to the next step, and the nucleic acids 71 ​​can be detected.

[0074] Additionally, due to their strong magnetization, magnetic beads 80 made of Fe-based amorphous metal form needle-like structures along the magnetic field lines when a magnetic field is applied. The needle-like structures increase the porosity of the aggregate of magnetic beads 80, making it easier to retain liquid 50 and carry over more of the reagent components to the next process, such as the eluate. However, by using the cartridge 100, the amount of liquid 50 carried over to the next process can be minimized.

[0075] Furthermore, in the cartridge 100 of this embodiment, the second flow path 21 preferably has a tapered shape that gradually decreases from the second inner diameter D2 toward the connection part 40. With this configuration, the size of the second flow path 21 gradually decreases, which makes it easier to introduce the liquid 50 into the container 30 and also makes it easier to bring the liquid 50 into contact with the inner wall 21a of the second flow path 21. This makes it easier to separate the liquid 50 and the magnetic beads 80.

[0076] In the cartridge 100 of this embodiment, the angle of the tapered shape is preferably 10° to 70°. With this configuration, the angle is formed within the above-described range, so that the liquid 50 can be brought into contact with the inner wall 21a of the second flow path 21. This makes it possible to easily separate the liquid 50 and the magnetic beads 80, and allows only the magnetic beads 80 to move to the first flow path 11.

[0077] In the cartridge 100 of this embodiment, the diameter of the first inner diameter D1 is preferably 1 mm to 2.5 mm. With this configuration, the diameter of the first inner diameter D1 is formed within the above-described range, so that the magnetic beads 80 and the liquid 50 can be separated and the magnetic beads 80 can pass through the first flow path 11.

[0078] Furthermore, in the cartridge 100 of this embodiment, a magnet 200 that generates a magnetic force is preferably arranged on the outside of the container 30 and is movable along the extension direction of the first tube section 10 and the second tube section 20. According to this configuration, the magnet 200 is arranged so as to be movable along the extension direction of the first tube section 10 and the second tube section 20, and therefore the magnetic force of the magnet 200 can be used to move the magnetic beads 80 in a desired direction.

[0079] In the cartridge 100 of this embodiment, the magnetic field gradient of the magnetic force is preferably 4 T / m to 237 T / m. With this configuration, since the magnetic force is in the above range, the magnetic beads 80 can be moved in a desired direction by the magnetic force.

[0080] In the cartridge 100 of this embodiment, the magnetic core 81 constituting the magnetic beads 80 is preferably made of an Fe-based amorphous metal. With this configuration, since the magnetic core 81 is made of an Fe-based amorphous metal, it is possible to improve the ability to follow changes in the direction of the magnetic force.

[0081] In the cartridge 100 of this embodiment, the saturation magnetization of the magnetic core 81 made of Fe-based amorphous metal is 50 Am 2 / kg or more. With this configuration, since the saturation magnetization is within the above range, it is possible to improve the ability to follow changes in the direction of magnetic force.

[0082] In the cartridge 100 of this embodiment, the size of the magnetic core 81 of the Fe-based amorphous metal is preferably such that the average particle diameter is 3 μm. With this configuration, since the diameter of the magnetic core 81 is the above-mentioned value, the nucleic acid 71 can be adsorbed to the magnetic beads 80.

[0083] In the cartridge 100 of this embodiment, the surface of the Fe-based amorphous metal magnetic core 81 is preferably coated with a silicon oxide film 82 having a thickness of 5 nm to 100 nm. With this configuration, the surface of the magnetic core 81 is coated with a silicon oxide film 82 having a thickness in the above range, so that the nucleic acid 71 can be adsorbed onto the magnetic beads 80.

[0084] Furthermore, the system 1000 for extracting, purifying, and separating nucleic acids 71 ​​of this embodiment includes a container 30 having a first tube section 10 provided with a first flow path 11 having a first inner diameter D1, and a second tube section 20 connected to the first tube section 10 via a connection section 40 and provided with a second flow path 21 having a second inner diameter D2 larger than the first inner diameter D1 and connected to the first flow path 11, a liquid 50 containing magnetic beads 80 introduced into the second flow path 21 of the container 30, a magnet 200 provided on the outside of the container 30 and causing the magnetic beads 80 to generate a magnetic force, and a magnet 200 and a second tube section 20 connected to the container 30 in the direction of extension of the first tube section 10 and the second tube section 20 of the container 30. and a moving mechanism 300 that changes the relative positional relationship between the magnet 200 and the first and second tube sections 10 and 20 of the container 30. The moving mechanism 300 changes the relative positional relationship between the magnet 200 and the first and second tube sections 10 and 20 of the container 30 from a state in which the magnet 200 corresponds to the second tube section 20, to a state in which the magnet 200 corresponds to the connection section 40, and then to a state in which the magnet 200 corresponds to the first tube section 10. This makes it possible to extract magnetic beads 80 that have bound to nucleic acids 71 ​​from the liquid 50 to the first tube section 10 side while the liquid 50 is held on the connection section 40 side of the second tube section 20.

[0085] According to this configuration, the moving mechanism 300 moves the liquid 50 containing the magnetic beads 80 introduced into the second flow path 21 toward the first flow path 11 by passing through the connection part 40 using magnetic attraction, and the movement speed of the liquid 50 is slowed down by the friction generated between the inner wall 21a of the second flow path 21 and the liquid 50. Therefore, even when the amount of liquid 50 contained is small, it is possible to efficiently extract and separate the magnetic beads 80 bound to the nucleic acids 71, and a group of multiple magnetic beads 80 containing a small amount of liquid 50 can be transferred to the next step. This makes it possible to prevent the liquid 50 from being carried over to the next step, and detect the nucleic acids 71.

[0086] In the nucleic acid 71 extraction, purification, and separation system 1000 of this embodiment, the moving mechanism 300 preferably moves the magnet 200 from the second tube section 20 side to the first tube section 10 side with respect to the container 30.

[0087] According to this configuration, the magnet 200 is moved from the second tube section 20 side to the first tube section 10 side with respect to the container 30, so that the magnetic beads 80 can be extracted and separated from the liquid 50 with a relatively simple configuration.

[0088] The nucleic acid extraction method of this embodiment includes a first tube section 10 provided with a first flow path 11 having a first inner diameter D1, and a second tube section 20 provided with a second flow path 21 connected to the first tube section 10 via a connection section 40, having a second inner diameter D2 larger than the first inner diameter D1 and communicating with the first flow path 11. The method includes the steps of: introducing a liquid 50 containing magnetic beads 80 into the second flow path 21 of the second tube section 20; and detecting a magnet 200 disposed outside the first tube section 10 and the second tube section 20, the magnet 200 being positioned on the outer side of the first tube section 10 and the second tube section 20, and detecting a magnetic bead 80 in the second flow path 21 of the second tube section 20. The method includes a step of changing the relative positional relationship in the direction from a state in which the magnet 200 corresponds to the second tube section 20 to a state in which the magnet 200 corresponds to the first tube section 10, and using the magnetic force acting between the magnetic beads 80 and the magnet 200 to pass the liquid 50 containing the magnetic beads 80 in the second flow path 21 through the connection section 40, and a step of extracting the magnetic beads 80 bound to the nucleic acids 71 ​​from the liquid 50 to the first tube section 10 side while retaining the liquid 50 on the connection section 40 side of the second tube section 20.

[0089] According to this method, the liquid 50 containing the magnetic beads 80 in the second flow path 21 is moved toward the first flow path 11 by magnetic attraction, and the movement speed of the liquid 50 is slowed down by the friction generated between the inner wall 21a of the second flow path 21 and the liquid 50. Therefore, it is possible to efficiently extract and separate the magnetic beads 80 bound to the nucleic acids 71 ​​even when the amount of liquid 50 contained is small, and a group of multiple magnetic beads 80 containing a small amount of liquid 50 can be transferred to the next step. This makes it possible to prevent the liquid 50 from being carried over to the next step, and allows the nucleic acids 71 ​​to be detected.

[0090] Modifications of the above-described embodiment will now be described.

[0091] As described above, the extraction of nucleic acids 71 ​​has been described, but it may also be applied to the extraction of antibodies, extracellular vesicles, cells, proteins, peptides, bacteria, viruses, algae, low molecular weight compounds, heavy metals, etc. [Explanation of symbols]

[0092] 10...first tube section, 10a...end section, 11...first flow path, 20...second tube section, 20a...end section, 21...second flow path, 21a...inner wall, 30...container, 40...connection section, 50...liquid, 60...third tube section, 61...third flow path, 71...nucleic acid, 80...magnetic beads, 81...magnetic core, 82...silicon oxide membrane, 100...cartridge, 200...magnet, 300...moving mechanism, 1000...extraction, purification, and separation system.

Claims

1. A cartridge for extracting nucleic acids using magnetic beads, a first pipe portion provided with a first flow path having a first inner diameter; and a second pipe portion communicated with the first pipe portion via a connection portion, the second pipe portion provided with a second flow path having a second inner diameter larger than the first inner diameter and communicating with the first flow path; and the second flow path of the second pipe portion has a tapered portion that gradually becomes smaller toward the connecting portion, A cartridge in which the magnetic beads can be removed from a liquid introduced into the cartridge to the first tube side by an external magnetic force, while the liquid is retained on the connection side of the second tube side.

2. A cartridge for extracting nucleic acids using magnetic beads, a first pipe portion provided with a first flow path having a first inner diameter; and a second pipe portion connected to the first pipe portion via a connection portion, the second pipe portion provided with a second flow path having a second inner diameter larger than the first inner diameter and communicating with the first flow path; a cleaning liquid provided corresponding to the second pipe portion; and the second flow path of the second pipe portion has a tapered portion that gradually becomes smaller toward the connecting portion, A cartridge in which the magnetic beads bound to nucleic acids from the washing solution can be removed to the first tube side by an external magnetic force, while the washing solution is retained on the connection side of the second tube part.

3. A cartridge according to claim 1 or claim 2, The angle formed between a line constituting the tapered portion and an extension of the first flow path is 10° to 70°.

4. 3. The cartridge of claim 2, A cartridge characterized in that an inlet for introducing a sample containing the nucleic acid into the cartridge is provided on the opposite side of the second tube section from the connection section, and an adsorption solution containing the cleaning solution and a chaotropic substance is provided between the inlet and the connection section.

5. 3. The cartridge of claim 2, A cartridge characterized in that a storage section for storing a mixture of an adsorption liquid containing a chaotropic substance and magnetic beads bound to nucleic acids is detachably attached to the other end of the second tube section opposite the connection section, and when attached, the storage section is connected to the second flow path of the second tube section so that the mixture can be introduced into the cartridge.

6. 6. The cartridge according to claim 4 or claim 5, A cartridge characterized in that an elution liquid for separating the nucleic acids bound to the magnetic beads and eluting them into a liquid is provided corresponding to the first tube portion side of the connection portion.

7. 7. The cartridge of claim 6, a cartridge characterized in that another connection part is provided on the opposite side of the first tube part to the connection part, and a third tube part having a third flow path communicating with the first flow path and having a third inner diameter larger than the first inner diameter is connected via the other connection part, and the eluate is provided corresponding to the third tube part.

8. 8. The cartridge according to claim 1, A cartridge, wherein the first inner diameter is 1 mm to 2.5 mm.

9. 9. The cartridge according to claim 1, A cartridge, wherein the magnetic beads are particles having an Fe-based amorphous metal core.

10. 10. The cartridge of claim 9, The saturation magnetization of the magnetic beads of the Fe-based amorphous metal is 50 Am 2 / kg or more.

11. 11. The cartridge according to claim 9 or claim 10, A cartridge in which the magnetic beads of the Fe-based amorphous metal have an average particle diameter of 3 μm.

12. 12. A cartridge according to any one of claims 9 to 11, The magnetic beads are particles having a core made of the Fe-based amorphous metal, the surfaces of which are coated with silicon oxide to a thickness of 5 nm to 100 nm.

13. a container including a first pipe portion provided with a first flow path having a first inner diameter, and a second pipe portion communicated with the first pipe portion via a connection portion, the second pipe portion provided with a second flow path having a second inner diameter larger than the first inner diameter and communicating with the first flow path; a liquid containing magnetic beads introduced into the second flow path of the container; a magnet provided outside the container to generate a magnetic force on the magnetic beads; a moving mechanism that varies a relative positional relationship between the magnet and the first and second pipe portions of the container in an extension direction of the first and second pipe portions of the container; Equipped with the second flow path of the second pipe portion has a tapered portion that gradually becomes smaller toward the connecting portion, A nucleic acid extraction, purification, and separation system in which the moving mechanism changes the relative positional relationship between the magnet and the first and second tube portions of the container from a state in which the magnet corresponds to the second tube portion, to a state in which the magnet corresponds to the connection portion, and then to a state in which the magnet corresponds to the first tube portion, thereby making it possible to remove the magnetic beads bound to nucleic acids from the liquid to the first tube portion side while the liquid is retained on the connection portion side of the second tube portion.

14. The extraction, purification, and separation system according to claim 13, An extraction, purification, and separation system, wherein the magnetic field gradient of the magnetic force is 4 T / m to 237 T / m.

15. The nucleic acid extraction, purification, and separation system according to claim 13 or 14, The moving mechanism moves the magnet from the second tube part side to the first tube part side with respect to the container.

16. a step of introducing a liquid containing magnetic beads into the second flow path of a first pipe section, the second pipe section being connected to the first pipe section via a connecting section and having a second flow path with a second inner diameter larger than the first inner diameter and a tapered portion that gradually becomes smaller toward the connecting section and communicates with the first flow path; a step of changing the relative positional relationship of magnets arranged outside the first tube portion and the second tube portion in the extending direction of the first tube portion and the second tube portion from a state in which the magnets correspond to the second tube portion to a state in which the magnets correspond to the first tube portion, and passing the magnetic beads in the second flow path through the connecting portion using a magnetic force acting between the magnetic beads and the magnets; removing the magnetic beads from the liquid to the first tube portion while the liquid is held at the connection portion of the second tube portion in an amount greater than the amount of liquid contained in the magnetic beads removed to the first tube portion; A nucleic acid extraction method comprising:

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