JP7643021B2 - Nucleic acid extraction instrument and method for producing same - Google Patents

Description

The present invention relates to an instrument for extracting nucleic acids contained in a sample.

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 excellent sensitivity and specificity.

In methods for detecting nucleic acids, a pretreatment step is performed to extract nucleic acids of pathogens, etc. from specimens such as whole blood, plasma, and throat swabs. This treatment involves complex steps that require skill, such as dissolving the pathogens in the specimen to release 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).

Patent No. 4477147 Patent No. 4911264 Patent No. 4379716 International Publication No. 2019/189897

The devices disclosed in Patent Documents 1 and 2 automate the nucleic acid extraction process, but are expensive and relatively large, making them unsuitable for portability.

The device disclosed in Patent Document 3 is a relatively low-cost, small, portable, planar device that is made by stacking two elastic bodies or an elastic body and a hard substrate, providing a gap inside to serve as a container or flow path for specimens or medicines, and pressing the device while changing its position to carry out the above-mentioned process.

Patent Document 4 discloses a device similar to that of Patent Document 3, but in this device, the soft base material and the soft film are sealed in a predetermined pattern, and the unsealed areas are used as storage sections and flow paths, making the device thinner and more portable than the device disclosed in Patent Document 3.

If a storage section or flow path is provided by forming an unsealed area, the heat used for the sealing process may reach this area and seal this area unintentionally, resulting in an inability to properly form the storage section or flow path and to properly extract nucleic acid.

The present invention was made in consideration of these problems, and aims to provide a nucleic acid extraction device that is made by sealing two films and can extract nucleic acids effectively.

One aspect of the present invention for solving the above problem is a nucleic acid extraction device that extracts nucleic acid from a specimen by being pressed by a roller that moves from one end to the other end , the device comprising: a first film in which recesses are formed to provide a plurality of storage sections capable of storing a chemical solution and a plurality of flow paths through which the chemical solution passes; and a second film that is sealed to the first film in a seal region excluding the storage sections and the flow paths of the first film ; the storage sections include, in order from 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 a waste liquid storage section; the flow paths include a flow path connecting the dissolution liquid storage section and the glass filter storage section, a flow path connecting the cleaning 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 specimen receiving section connected to a flow path connecting the glass filter storage section and the glass filter storage section, for introducing a specimen; and a restricting section for restricting movement of the chemical liquid by weakly sealing the first film and the second film at the other end of each of the dissolution liquid storage section, the cleaning liquid storage section, the elution liquid storage section and the temporary storage section, wherein 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 so that the weak seals of the restricting sections provided at the other end of each are peeled off, and the specimen introduced from the specimen receiving section and the chemical liquid stored in each storage section are moved to the glass filter storage section, and the chemical liquid that has passed through the glass filter storage section is moved to the waste liquid storage section, and after the roller has passed 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 at the other end of the temporary storage section, and the nucleic acid elution liquid stored in the temporary storage section is moved to the nucleic acid elution liquid storage section .

Another aspect of the present invention is a method for manufacturing the above-mentioned nucleic acid extraction instrument, comprising the steps of forming a recess in a first film, housing a chemical solution in the recess, and, after housing the chemical solution in the recess, sealing a second film to an area of the first film excluding the recess.

According to the present invention, in a nucleic acid extraction device constructed by sealing two films, the container and the chemical solution are formed by recesses provided in the film, making it difficult for the container and the chemical solution to be sealed, and allowing for optimal nucleic acid extraction.

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.

(Embodiment)
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.

In addition, the storage section or flow path is provided with one or more restriction sections that weakly seal the first film 101 and the second film 102 to restrict the movement of the drug solution.

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

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

The nucleic acid extraction instrument 100 includes a sample receiving section 31 that is configured to receive a sample (not shown). In the sample receiving section 31, a filter 32 that filters 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.

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

Here we explain how to extract nucleic acid.

First, the sample is dropped into the sample receiving section 31. The sample from which unnecessary substances have been filtered out flows into the flow path 11.

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 table, and sandwiching and pressing the nucleic acid extraction instrument 100 between a roller whose axes are aligned in the vertical direction of FIG. 1 and the table while moving the roller from left to right. Alternatively, the nucleic acid extraction instrument 100 may be sandwiched between two rollers and pressed in the same manner.

As the intersection of the roller axis and the solid arrow in Figure 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 acid is released. The lysis liquid containing the nucleic acid flows from the flow path 11 to the glass filter storage section 51 and passes through the glass filter 52, and the nucleic acid is adsorbed by the glass filter 52. The lysis liquid that has passed through the glass filter 52 passes through the flow path 15, storage section 5, and flow path 16 and is stored in the waste liquid storage section 61.

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. After that, 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 chemical liquid and the flow rate associated with the movement of the pressing position.

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 restricting section 24 peels off, and the eluate flows through the flow path 14 into the glass filter storage section 51.

As the intersection of the roller axis and the solid arrow shown in Figure 1 moves from P4 to P5, the eluate passes through the glass filter 52, and the nucleic acid adsorbed to the glass filter 52 is eluted into the eluate. The eluate from which the nucleic acid has eluted passes through the flow path 15 and is stored in the storage section 5.

As the intersection of the roller axis and the solid arrow shown in Figure 1 moves from P5 to P6, the flow path 16 is pressed and closed. The eluate contained in the storage section 5 is no longer able to pass through the flow path 16, the internal pressure increases, the weak seal of the regulating 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 with the roller, only the desired eluate can be contained in the outlet 41 in the final stage.

The eluate is then removed from outlet 41 and used for testing, etc.

The above explanation is a schematic explanation of the principle, and as long as the eluate from which the nucleic acids have eluted can be made to flow into outlet 41, the timing at which the drug solution actually moves through each flow path may vary slightly. Also, 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, etc.

The nucleic acid extraction instrument 100 is manufactured by forming recesses in the first film 101 to serve as storage sections and flow paths, and then storing the filter 32, glass filter 52, and chemical liquids such as dissolving liquid, cleaning liquid, and elution liquid in the respective recesses, and then sealing with the second film 102. The recesses make it difficult for the storage sections and flow paths to be sealed, so the storage sections and flow paths can be formed favorably. In addition, the storage sections and flow paths can be formed not simply as unsealed areas, but as spaces that can store and pass chemical liquids, which is preferable. In addition, the nucleic acid extraction instrument 100 can be manufactured in 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.

For example, each flow path preferably 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, in order to allow the drug solution to move efficiently and to reduce residual amounts. Note that, depending on the amount of drug solution to pass through, there may be flow paths that fall outside this range, but in order to prevent sealing, it is preferable that the depth be 0.1 mm or more.

The adhesive strength of the weak seal at 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 sealing area is preferably 20 times or more, more preferably 40 times or more, and even more preferably 50 times or more, of the adhesive strength at each restricting portion in order to suppress peeling. For example, the strong seal can be performed under sealing conditions of 190°C, 0.4 MPa, and 2 seconds, and the weak seal can be performed under sealing conditions of 160°C, 0.4 MPa, and 2 seconds.

It is preferable that each storage section has a shape in which the width narrows and the depth decreases in the outflow direction to which the flow path is connected. This makes it easier for the stored medicinal liquid to flow out and reduces the amount of remaining medicinal liquid. The shape of the storage section may be other shapes depending on the application and the amount of medicinal liquid to be stored. Like the flow path, each storage section is preferably 0.1 mm or more deep to prevent it from being sealed.

The filter 32 may be selected appropriately depending on the type of sample, but for example, a blood cell separation filter (Whatman, Fusion 5) can be used.

The glass filter 52 may be, for example, Advantech's GA-100. 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.

The first film 101 and the second film 102 are preferably 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 favorably. Alternatively, the first film 101 and the second film 102 are preferably 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, favorable ease of deformation and durability are easily obtained.

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.

The first film 101 and the second film 102 are, for example, laminated films obtained by laminating nylon and linear low-density polyethylene by 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.

The waste liquid storage section 61, like the other storage sections, 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 crushed, and that 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 it can preferably store unnecessary chemical liquid. In addition, a water-absorbent resin or the like may be placed in the waste liquid storage section 61 in advance to suppress backflow.

As described above, the nucleic acid extraction instrument of the present invention forms the container and the chemical solution using recesses provided in the film, which makes it difficult for the container and the chemical solution to be sealed, allowing for suitable nucleic acid extraction. In addition, since the container and the chemical solution can be sealed after being placed in the container, manufacturing is also easy. The present invention can be applied not only to nucleic acid extraction, but also to instruments for processing various biological samples.

The present invention is useful for portable nucleic acid extraction devices, etc.

REFERENCE SIGNS LIST 1 Dissolution liquid storage section 2 Washing liquid storage section 3 Washing liquid storage section 4 Elution liquid storage section 5 Storage section 11, 12, 13, 14, 15, 16, 17 Flow path 21, 22, 23, 24, 25 Restriction section 31 Sample receiving section 32 Filter 41 Extraction port 51 Glass filter storage section 52 Glass filter 61 Waste liquid storage section 100 Nucleic acid extraction device 101 First film 102 Second film

Claims (6)

A nucleic acid extraction device for extracting nucleic acid from a specimen by pressing the specimen 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 weak seal of the regulating section provided on the other end side of the temporary storage section is peeled off by pressing the temporary storage section while the roller is blocking the flow path connecting the temporary storage section and the waste liquid storage section by pressing the temporary storage section after the roller has passed through the glass filter storage section, and the nucleic acid elution liquid stored in the temporary storage section is moved to the nucleic acid elution liquid storage section . The nucleic acid extraction device according to claim 1, wherein any one of the flow 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. 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. The nucleic acid extraction instrument according to claim 1 , wherein any one of said storage sections has a shape that narrows in width and decreases in depth in an outflow direction of the stored chemical solution. 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. 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.

Images