Nucleic acid extraction instrument and method for producing same

Abstract

To provide a nucleic acid extractor that is made up of sealed two films and allows for suitable extraction of nucleic acids.SOLUTION: A nucleic acid extractor for extracting nucleic acids from a sample, includes a first film that is provided with a recess, the recess constituting one or more housings capable of storing a chemical solution and one or more channels for allowing the chemical solution to pass through, and a second film that is sealed with the first film in a seal region of the first film excluding the housings and channels. A method for producing a nucleic acid extractor includes the steps of: forming a recess on a first film; storing a chemical solution in the recess; and, following the chemical solution-storage step, sealing a second film in a region of the first film excluding the recess.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an instrument for extracting nucleic acid contained in a sample. [Background technology]

[0002] Tests for infectious diseases can be broadly divided into methods that use antigen-antibody reactions and methods that detect the nucleic acid of pathogens. Methods that detect nucleic acid have superior sensitivity and specificity.

[0003] In a method for detecting nucleic acid, a pretreatment is performed to extract nucleic acids of pathogens, etc. from specimens such as whole blood, plasma, and throat swabs. This treatment involves complex and skilled steps of dissolving pathogens in the specimen to liberate the nucleic acids, specifically adsorbing the nucleic acids to a carrier, washing the carrier, and then eluting the nucleic acids. Therefore, devices and instruments for easily performing such nucleic acid extraction have been proposed (Patent Documents 1 to 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4477147 [Patent Document 2] Patent No. 4911264 [Patent Document 3] Patent No. 4379716 [Patent Document 4] International Publication No. 2019 / 189897 Summary of the Invention [Problem to be solved by the invention]

[0005] The devices disclosed in Patent Documents 1 and 2 automate the nucleic acid extraction process, but are expensive and relatively large and not suitable for portability.

[0006] The device disclosed in Patent Document 3 is a relatively low-cost, small, portable, planar device that is made by stacking two elastomers or an elastomer and a hard substrate, and providing spaces inside to serve as storage compartments and flow paths for specimens and chemicals. The above-mentioned process can be carried out by pressing the device while changing its position.

[0007] Patent Document 4 discloses a device similar to that of Patent Document 3, but in this device, the soft substrate and the soft film are sealed in a predetermined pattern, and the unsealed areas are used as a storage section and a flow path, making the device thinner and more portable than the device disclosed in Patent Document 3.

[0008] When a storage section or flow path is provided by forming an unsealed area, the heat used for the sealing process may extend to this area, causing this area to be sealed unintentionally, which may result in the storage section or flow path not being formed properly and making it difficult to extract nucleic acid properly.

[0009] The present invention has been made in view of the above problems, and has an object to provide a nucleic acid extraction instrument which is constructed by sealing two sheets of film and is capable of suitably extracting nucleic acid. [Means for solving the problem]

[0010] One aspect of the present invention for solving the above problem is to By pressing with a roller moving from one end to the other A nucleic acid extraction device for extracting nucleic acid from a specimen, the device having a recess capable of accommodating a liquid medicine. multiple The container and the drug solution pass through multiple The device includes a first film having a flow path, and a second film sealed to the first film in a sealing region excluding the storage portion and the flow path of the first film. The storage sections include, in order from one end to the other end, a lysis liquid storage section, a washing liquid storage section, an elution liquid storage section, a glass filter storage section, a temporary storage section, and a nucleic acid elution liquid storage section, and a waste liquid storage section. The flow paths include a flow path connecting the lysis liquid storage section and the glass filter storage section, a flow path connecting the washing liquid storage section and the glass filter storage section, a flow path connecting the elution liquid storage section and the glass filter storage section, a flow path connecting the glass filter storage section and the temporary storage section, a flow path connecting the temporary storage section and the waste liquid storage section, and a flow path connecting the temporary storage section and the nucleic acid elution liquid storage section. A sample receiving section is provided, connected to the flow path connecting the lysis liquid storage section and the glass filter storage section, for introducing a sample, and at the other end sides of the lysis liquid storage section, the washing liquid storage section, the elution liquid storage section, and the temporary storage section, a restricting section is provided by weakly sealing the first film and the second film to restrict movement of the drug solution, and as the roller moves from one end to the other end, each of the storage sections, the dissolution liquid storage section, the cleaning liquid storage section and the elution liquid storage section, is pressed in this order to peel off the weak seals of the restricting sections provided on the other end sides of each storage section, the sample introduced from the sample receiving section and the drug solution stored in each storage section move to the glass filter storage section, the drug solution that has passed through the glass filter storage section moves to the waste liquid storage section, and after the roller passes through the glass filter storage section, the roller presses the temporary storage section while blocking the flow path connecting the temporary storage section and the waste liquid storage section, thereby peeling off the weak seal of the restricting section provided on the other end side of the temporary storage section, and the nucleic acid elution liquid stored in the temporary storage section moves to the nucleic acid elution liquid storage section. This is a nucleic acid extraction instrument.

[0011] Another aspect of the present invention includes a step of forming a recess in a first film, a step of housing a medicinal solution in the recess, and a step of sealing a second film to an area of ​​the first film excluding the recess after the step of housing the medicinal solution in the recess. The above A method for manufacturing a nucleic acid extraction instrument. Effect of the Invention

[0012] According to the present invention, in a nucleic acid extraction device constructed by sealing two films, a container and a chemical solution are formed by recesses provided in the films, so that the container and the chemical solution are less likely to be sealed, enabling nucleic acid extraction to be performed suitably. [Brief description of the drawings]

[0013] [Figure 1] 1 is a schematic plan view and a schematic partial cross-sectional view of a nucleic acid extraction instrument according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] (Embodiment) FIG. 1 shows a schematic plan view and a schematic partial cross-sectional view taken along line A-A' of a nucleic acid extraction instrument according to one embodiment of the present invention. The nucleic acid extraction instrument 100 is formed by sealing a first film 101 and a second film 102 made of a soft material together. One or more recesses are formed in the first film 101 by vacuum molding or the like. These recesses form a reservoir section and a flow path for storing a chemical solution. The second film 102 is sealed to the first film in a sealing region excluding the reservoir section and the flow path.

[0015] The container or the flow path is provided with one or more restriction sections where first film 101 and second film 102 are weakly sealed to restrict movement of the drug solution.

[0016] The nucleic acid extraction instrument 100 includes, for example, storage units 1, 2, 3, 4, 5, 41, 51, and 61. The storage unit 1 is a lysis liquid storage unit that previously contains a lysis buffer for dissolving cells, bacteria, and viruses in a specimen. The storage units 2 and 3 are cleaning liquid storage units that previously contain a cleaning liquid (e.g., ethanol) for washing the carrier. The storage unit 4 is an elution liquid storage unit that previously contains an elution buffer for eluting nucleic acid from the carrier. The storage unit 5 temporarily contains a chemical solution that has passed through the carrier. The storage unit 41 is an outlet that contains an elution liquid containing nucleic acid and is provided with a structure for removal (not shown). The storage unit 51 is a glass filter storage unit that previously contains a glass filter 52 as a carrier. The storage unit 61 is a waste liquid storage unit that contains unnecessary waste liquid.

[0017] The nucleic acid extraction instrument 100 includes, for example, flow paths 11, 12, 13, 14, 15, 16, and 17. Flow path 11 connects the lysis liquid storage section 1 to the glass filter storage section 51. Flow path 12 connects the cleaning liquid storage section 2 to the cleaning liquid storage section 3. Flow path 13 connects the cleaning liquid storage section 3 to the glass filter storage section 51. Flow path 14 connects the elution liquid storage section 4 to the glass filter storage section 51. Flow path 15 connects the glass filter storage section 51 to the storage section 5. Flow path 16 connects the storage section 5 to the waste liquid storage section 61. Flow path 17 connects the storage section 5 to the outlet 41.

[0018] The nucleic acid extraction instrument 100 includes a sample receiving section 31 having a structure for receiving a sample (not shown). In the sample receiving section 31, a filter 32 for filtering out unwanted substances contained in the sample is sandwiched between the first film 101 and the second film, and the sample passes through the filter 32 and flows into the flow path 11.

[0019] The nucleic acid extraction instrument 100 includes, for example, restriction units 21, 22, 23, 24, and 25. The restriction unit 21 is provided in the dissolution liquid storage unit 1. The restriction unit 22 is provided in the cleaning liquid storage unit 2. The restriction unit 23 is provided in the cleaning liquid storage unit 3. The restriction unit 24 is provided in the elution liquid storage unit 4. The restriction unit 25 is provided in the storage unit 5.

[0020] Here, a method for nucleic acid extraction will be described.

[0021] First, the specimen is dropped and injected into the specimen receiving portion 31. The specimen from which unnecessary substances have been filtered out flows into the flow channel 11.

[0022] The pressing position is gradually moved from the left end of the nucleic acid extraction instrument 100 shown in Fig. 1 toward the right side in parallel with the direction of the solid arrow. This is done, for example, by placing the nucleic acid extraction instrument 100 on a flat stand, and moving the roller from left to right while pressing the nucleic acid extraction instrument 100 between the stand and a roller whose axes are aligned in the vertical direction of Fig. 1. Alternatively, the nucleic acid extraction instrument 100 may be sandwiched between two rollers and pressed in the same manner.

[0023] As the intersection of the roller axis and the solid arrow shown in FIG. 1 moves from P1 to P2, the lysis liquid storage section 1 is pressed, the internal pressure of the stored lysis liquid increases, the weak seal of the restriction section 21 peels off, and the lysis liquid flows into the flow path 11. The lysis liquid and the specimen are mixed in the flow path 11, the cells, bacteria, and viruses contained in the specimen are dissolved by the lysis liquid, and the nucleic acids are released. The lysis liquid containing the nucleic acids flows from the flow path 11 to the glass filter storage section 51, passes through the glass filter 52, and the nucleic acids are adsorbed to the glass filter 52. The lysis liquid that has passed through the glass filter 52 passes through the flow path 15, the storage section 5, and the flow path 16, and is stored in the waste liquid storage section 61.

[0024] As the intersection of the roller axis and the solid arrow shown in FIG. 1 moves from P2 to P3, the cleaning liquid storage section 2 is pressed, the internal pressure of the stored cleaning liquid increases, the weak seal of the regulating section 22 peels off, and the cleaning liquid flows through the flow path 12 to the cleaning liquid storage section 3. Thereafter, the cleaning liquid storage section 3 is pressed, the internal pressure of the stored cleaning liquid increases, the weak seal of the regulating section 23 peels off, and the cleaning liquid flows through the flow path 13 to the glass filter storage section 51, and the glass filter 52 is cleaned. The cleaning liquid that has passed through the glass filter 52 passes through the flow path 15, the storage section 5, and the flow path 16 and is stored in the waste liquid storage section 61. By storing the same chemical liquid in multiple storage sections connected along the pressing movement direction, as in the cleaning liquid storage sections 2 and 3, it is possible to easily adjust the total amount of the chemical liquid and the flow rate associated with the movement of the pressing position.

[0025] As the intersection of the roller axis and the solid arrow shown in Figure 1 moves from P3 to P4, the eluate storage section 4 is pressed, the internal pressure of the stored eluate increases, the weak seal of the regulating section 24 peels off, and the eluate flows through the flow path 14 into the glass filter storage section 51.

[0026] 1 moves from P4 to P5, the eluate passes through glass filter 52, and the nucleic acid adsorbed to glass filter 52 is eluted into the eluate. The eluate from which the nucleic acid has been eluted passes through flow channel 15 and is stored in storage unit 5.

[0027] 1 moves from P5 to P6, the flow path 16 is pressed and closed. The eluate contained in the storage section 5 cannot pass through the flow path 16, the internal pressure increases, the weak seal of the restricting section 25 peels off, and the eluate flows through the flow path 17 to the outlet 41. In this way, by moving the roller in the direction from P1 to P6 while applying a pressing force by the roller, only the desired eluate can be contained in the outlet 41 in the final stage.

[0028] Thereafter, the eluate is taken out from the outlet 41 and used for testing or the like.

[0029] The above explanation is a schematic explanation of the principle, and the timing at which the drug solution actually moves through each flow path may vary slightly as long as the eluate from which the nucleic acid has been eluted can be made to flow into outlet 41. Furthermore, as long as the drug solution can be moved in a similar manner, the containers 1, 2, 3, 4, 5, etc. may be pressed in order with a tool other than a roller, a finger, or the like.

[0030] The nucleic acid extraction instrument 100 is manufactured by forming recesses in the first film 101 to become a storage section, a flow path, etc., and storing the filter 32, the glass filter 52, and chemical liquids such as a dissolving liquid, a cleaning liquid, and an elution liquid in the respective predetermined recesses, and then sealing with the second film 102. The recesses make it difficult for the storage section and the flow path to be sealed, so that the storage section and the flow path can be preferably formed. In addition, the storage section and the flow path can be preferably formed as a space that can store and pass the chemical liquid, rather than simply forming the storage section and the flow path as an unsealed area. In addition, the nucleic acid extraction instrument 100 can be manufactured by a simple process, compared to the conventional nucleic acid extraction instrument, which requires a process of bonding two films together and then injecting each chemical liquid into each storage section.

[0031] For example, each flow path preferably has a width of 0.5 mm to 3 mm and a depth of 0.1 mm to 2.0 mm in order to allow the liquid drug to move appropriately and to suppress residual amounts. Note that, although some flow paths may be outside this range depending on the planned amount of liquid drug to pass through, the depth is preferably 0.1 mm or more in order to prevent sealing.

[0032] The adhesive strength of the weak seal in each restricting portion between the first film 101 and the second film 102 is preferably 0.1 N / 15 mm width or more and 1 N / 15 mm width or less in order to have a certain degree of strength while favorably causing peeling. Also, the adhesive strength of the strong seal in the seal region is preferably 20 times or more, more preferably 40 times or more, and even more preferably 50 times or more, of the adhesive strength in each restricting portion in order to suppress peeling. For example, the strong seal is performed under sealing conditions of 190°C, 0.4 MPa, and 2 seconds, and the weak seal is performed under sealing conditions of 160°C, 0.4 MPa, and 2 seconds.

[0033] Each storage section preferably has a shape in which the width narrows and the depth decreases toward the outflow direction to which the flow path is connected. This allows the stored liquid medicine to easily flow out and reduces the amount of remaining liquid medicine. The shape of the storage section may be other shapes depending on the purpose and the amount of liquid medicine to be stored. Each storage section, like the flow path, preferably has a depth of 0.1 mm or more to prevent it from being sealed.

[0034] The filter 32 may be appropriately selected depending on the type of specimen, and for example, a blood cell separation filter (Whatman, Fusion5) may be used.

[0035] For example, Advantech's GA-100 can be used as the glass filter 52. Note that the nucleic acid extraction instrument 100 may be provided with a container that contains magnetic beads instead of the glass filter container 51 that contains the glass filter 52.

[0036] It is preferable that the first film 101 and the second film 102 are made of a material having a loop stiffness of, for example, 120 mN / 15 mm width or less. By using such a material, the first film 101 and the second film 102 are easily deformed by pressure, and the drug solution can be moved suitably. Alternatively, it is preferable that the first film 101 and the second film 102 are made of a soft material having a thickness of, for example, 50 μm to 500 μm, or 200 μm to 300 μm. By using such a material, it is easy to obtain suitable ease of deformation and durability.

[0037] The first film 101 and the second film 102 may be a single layer film or a laminated film. The first film 101 and the second film 102 may be the same film or different films. In the case of a laminated film, the film may be a film manufactured by a co-extrusion method or a film manufactured by a lamination method. The sealing layer may be a polymer material whose main raw material is polyethylene or polypropylene, or an easy-peel material made of a mixture of multiple resins.

[0038] The first film 101 and the second film 102 are, for example, laminated films obtained by laminating nylon and linear low-density polyethylene using a co-extrusion method, and films with a thickness of 70 μm (loop stiffness 22 mN / 15 mm width), 120 μm (loop stiffness 28 mN / 15 mm width), 150 μm (loop stiffness 30 mN / 15 mm width), or 180 μm (loop stiffness 116 mN / 15 mm width) can be suitably used.

[0039] Like the other storage sections, the waste liquid storage section 61 is provided by forming a recess in the first film 101, but it is preferable that the first film 101 and the second film 102 are sealed with the recess pressed down, and the amount of air in the waste liquid storage section 61 is small and the volume is small when the nucleic acid extraction instrument 100 is not in use. As a result, when the nucleic acid extraction instrument 100 is in use, the volume of the waste liquid storage section 61 increases as the chemical liquid flows in, so that unnecessary chemical liquid can be suitably stored. Also, a water-absorbent resin or the like may be stored in the waste liquid storage section 61 in advance to suppress backflow.

[0040] As described above, the nucleic acid extraction instrument according to the present invention is suitable for nucleic acid extraction because the container and the chemical solution are formed by the recesses provided in the film, making it difficult for the container and the chemical solution to be sealed. In addition, since the container and the chemical solution can be sealed after being placed in the container, the instrument is easy to manufacture. The present invention can be applied not only to nucleic acid extraction, but also to instruments for various biological sample processing. [Industrial Applicability]

[0041] The present invention is useful for a portable nucleic acid extraction instrument and the like. [Explanation of symbols]

[0042] 1. Dissolving liquid storage section 2 Cleaning fluid storage section 3 Cleaning fluid storage section 4. Elution fluid storage section 5. Storage section 11, 12, 13, 14, 15, 16, 17 Flow path 21, 22, 23, 24, 25 Regulatory Department 31 Sample Receiving Section 32 Filters 41 Outlet 51 Glass filter storage section 52 Glass Filter 61 Waste liquid storage section 100 Nucleic acid extraction equipment 101 1st film 102 2nd Film

Claims

1. A nucleic acid extraction device for extracting nucleic acid from a specimen by pressing with a roller that moves from one end to the other end, a first film having recesses formed therein to provide a plurality of storage sections capable of storing drug solutions and a plurality of flow paths through which the drug solutions pass; a second film sealed to the first film at a sealing region excluding the storage portion and the flow path of the first film, The storage sections include, in order from the one end to the other end, a dissolution liquid storage section, a cleaning liquid storage section, an elution liquid storage section, a glass filter storage section, a temporary storage section, and a nucleic acid elution liquid storage section, and also a waste liquid storage section; the flow paths include a flow path connecting the lysis liquid storage unit and the glass filter storage unit, a flow path connecting the cleaning liquid storage unit and the glass filter storage unit, a flow path connecting the elution liquid storage unit and the glass filter storage unit, a flow path connecting the glass filter storage unit and the temporary storage unit, a flow path connecting the temporary storage unit and the waste liquid storage unit, and a flow path connecting the temporary storage unit and the nucleic acid elution liquid storage unit, a specimen receiving section is provided, the specimen receiving section being connected to a flow path connecting the dissolution liquid storage section and the glass filter storage section, and into which a specimen is introduced; a restricting section for restricting movement of the chemical solution by weakly sealing the first film and the second film, provided on the other end side of each of the dissolution liquid storage section, the cleaning liquid storage section, the elution liquid storage section and the temporary storage section; As the roller moves from the one end to the other end, each of the storage sections, the dissolution liquid storage section, the cleaning liquid storage section, and the elution liquid storage section, is pressed in this order, and the weak seals of the restriction sections provided on the other end sides of each of the storage sections are peeled off, the specimen introduced from the specimen receiving section and the chemical liquid stored in each of the storage sections move to the glass filter storage section, and the chemical liquid that has passed through the glass filter storage section moves to the waste liquid storage section, a nucleic acid extraction apparatus, characterized in that after the roller has passed through the glass filter storage section, the roller presses the temporary storage section while blocking the flow path connecting the temporary storage section and the waste liquid storage section, thereby peeling off the weak seal of the regulating section provided on the other end side of the temporary storage section, and the nucleic acid elution liquid stored in the temporary storage section moves to the nucleic acid elution liquid storage section.

2. 2. The nucleic acid extraction instrument according to claim 1, wherein any one of the channels has a width of 0.5 mm or more and 3 mm or less and a depth of 0.1 mm or more and 2.0 mm or less.

3. 3. The nucleic acid extraction instrument according to claim 1, wherein an adhesive strength between the first film and the second film at the restricting portion is 1 N / 15 mm width or less, and an adhesive strength in the sealing region is 20 times or more the adhesive strength at the restricting portion.

4. 4. The nucleic acid extraction instrument according to claim 1, wherein any one of the storage sections has a shape that narrows in width and decreases in depth in an outflow direction of the stored chemical solution.

5. The nucleic acid extraction instrument according to any one of claims 1 to 4, wherein the first film and the second film are made of a soft material having a thickness of 50 μm to 500 μm, or a material having a loop stiffness of 120 mN / 15 mm width or less.

6. forming a recess in a first film; storing a chemical solution in the recess; The method for producing a nucleic acid extraction instrument according to any one of claims 1 to 5, further comprising the step of sealing a second film to an area of ​​the first film excluding the recess after the step of storing a chemical solution in the recess.

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