Nucleic acid extraction containers and nucleic acid extraction methods

Abstract

In the nucleic acid extraction container (160), the filter section (166) has a hydrophilic filter (168), a biomaterial collection area (166a), and a sample permeation area (166b). The filter (168) collects nucleic acid-containing biomaterial from the sample. The biomaterial collection area (166a) collects biomaterial on the filter (168). The sample permeation area (166b) allows the sample that has passed through the filter to pass through. A sample inlet (174) is connected to the biomaterial collection area (166a). An air vent (178) is connected to the biomaterial collection area (166a). A sample outlet (176) is connected to the sample permeation area (166b). The air vent (178) is configured to be openable and closable relative to the outside.

Description

Technical Field

[0001] This invention relates to nucleic acid extraction containers and nucleic acid extraction methods. Background Technology

[0002] Genetic testing is widely used in various medical fields, for the identification of crops and pathogenic microorganisms, for food safety evaluation, and even for the detection of pathogenic viruses and various infectious diseases. To detect trace amounts of nucleic acids as genes with high sensitivity, methods are known for analyzing substances obtained by amplifying a portion of the nucleic acid. Among these, polymerase chain reaction (PCR) is a promising technique for selectively amplifying a portion of extremely small amounts of nucleic acid collected from organisms. To perform PCR, nucleic acids need to be extracted from the biological sample.

[0003] As an example of this type of genetic testing, there are known techniques for analyzing the presence of DNA (referred to as environmental DNA) from a specific organism using PCR to investigate whether it can survive in environments such as water, soil, and air (see, for example, Non-Patent Documents 1-3). For this analysis, the Environmental DNA Society has developed the "Experimental Manual for Environmental DNA Surveys" and recommends this method (hereinafter referred to as the Society's Standard Method) as the standard method for qualitative and quantitative determination (see Non-Patent Document 4). In the Society's Standard Method, river water or seawater is collected using a beaker, filtered, and then DNA is extracted.

[0004] (Existing technical literature)

[0005] (Patent Documents)

[0006] Non-patent literature 1: Hideyuki Doi et al., Freshwater Biology (2017) 62, 30-39

[0007] Non-patent literature 2: PLOS ONE | DOI:10.1371 / journal.pone.0149786 March 2, 2016

[0008] Non-patent literature 3: PLOS ONE | DOI:10.1371 / journal.pone.0142008November 4,2015

[0009] Non-patent document 4: "Environmental DNA Survey and Experiment Manual (ver 2.1)", Environmental DNA Society, Internet <URL: http: / / ednasociety.org / eDNA_manual_ver2_1_3.pdf> Summary of the Invention

[0010] (The problem the invention aims to solve)

[0011] The standardized method, which yields relatively accurate measurement results, is considered an efficient method (the proportion of DNA extracted from the environment that is ready for amplification such as PCR). However, this method has several drawbacks, including the multiple and time-consuming steps involved in DNA extraction from the sample, the need for extraction skills, significant operator bias in measurement results, and the large number and high cost of necessary components, equipment, and reagents. These problems exist not only in the extraction of environmental DNA but also in the extraction of nucleic acids used to identify microorganisms and viruses.

[0012] The present invention was made in view of this situation, and one of its objectives is to provide a technique for extracting nucleic acids from samples simply, quickly and at low cost.

[0013] (The measures taken to solve the problem)

[0014] One aspect of the present invention is a nucleic acid extraction container. This nucleic acid extraction container has:

[0015] The filter section includes a hydrophilic filter, a biomaterial collection area, and a sample passage area, wherein the filter collects biomaterial containing nucleic acids from the sample, the biomaterial collection area collects biomaterial on the filter, and the sample passage area allows the sample that has passed through the filter to pass through.

[0016] The sample injection port is connected to the biomaterial collection area;

[0017] An air vent, which connects to the biological material collection area; and

[0018] The sample outlet is connected to the sample permeation area.

[0019] The air vent is configured to be openable and closed relative to the outside.

[0020] Another aspect of the present invention is a nucleic acid extraction method. This nucleic acid extraction method includes:

[0021] A process of collecting nucleic acid-containing biological material on a filter by passing a sample through a filter section of a hydrophilic filter;

[0022] The process of injecting nucleic acid extraction solution into the filter section and extracting nucleic acid on the filter; and

[0023] The process of recovering the solution containing the extracted nucleic acid from the filter section.

[0024] Another aspect of the present invention is also a nucleic acid extraction method. This nucleic acid extraction method includes:

[0025] A process of collecting nucleic acid-containing biological material on a filter by passing a sample through a filter section of a hydrophilic filter;

[0026] The process of injecting nucleic acid extraction solution into the filter section and extracting nucleic acid on the filter;

[0027] The process of moving the solution containing the extracted nucleic acid from the filter into a flow path connected to the filter section; and

[0028] The process of dispensing a solution containing nucleic acids by injecting air into the flow path.

[0029] (The effect of the invention)

[0030] According to the present invention, nucleic acids can be extracted from samples simply, quickly, and at low cost. Attached Figure Description

[0031] Figure 1 (a) and Figure 1 (b) is a diagram illustrating the nucleic acid extraction container of the first embodiment of the present invention.

[0032] Figure 2 for Figure 1 (a) shows the AA cross-sectional view of the nucleic acid extraction container.

[0033] Figure 3 for Figure 1 (a) shows a BB cross-sectional view of the nucleic acid extraction container.

[0034] Figure 4 for Figure 1 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0035] Figure 5 This is a diagram illustrating a modified example 1 of a nucleic acid extraction container.

[0036] Figure 6 This is a diagram illustrating a modified example 2 of a nucleic acid extraction container.

[0037] Figure 7 This diagram illustrates the flow of a sample through the filter section.

[0038] Figure 8 This diagram illustrates the extraction of nucleic acid using nucleic acid extraction solution on a filter.

[0039] Figure 9 (a) and Figure 9 (b) is a diagram illustrating the nucleic acid extraction container of the second embodiment of the present invention.

[0040] Figure 10 for Figure 9 (a) shows the AA cross-sectional view of the nucleic acid extraction container.

[0041] Figure 11 for Figure 9 (a) shows a BB cross-sectional view of the nucleic acid extraction container.

[0042] Figure 12 for Figure 9 (a) shows a CC cross-sectional view of the nucleic acid extraction container.

[0043] Figure 13 for Figure 9 (a) shows a DD cross-sectional view of the nucleic acid extraction container.

[0044] Figure 14 for Figure 9 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0045] Figure 15 (a) and Figure 15 (b) is a diagram illustrating the nucleic acid extraction container of the third embodiment of the present invention.

[0046] Figure 16 for Figure 15 (a) shows the AA cross-sectional view of the nucleic acid extraction container.

[0047] Figure 17 for Figure 15 (a) shows an EE cross-sectional view of the nucleic acid extraction container.

[0048] Figure 18 for Figure 15 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0049] Figure 19 The diagram is intended to schematically illustrate the presence of a nucleic acid-containing solution in region C of the third flow path.

[0050] Figure 20 (a) and Figure 20 (b) is a diagram illustrating the nucleic acid extraction container of the fourth embodiment of the present invention.

[0051] Figure 21 for Figure 20 (a) shows the AA cross-sectional view of the nucleic acid extraction container.

[0052] Figure 22 for Figure 20 (a) shows a BB cross-sectional view of the nucleic acid extraction container.

[0053] Figure 23 for Figure 20 (a) shows a CC cross-sectional view of the nucleic acid extraction container.

[0054] Figure 24 for Figure 20 (a) shows a DD cross-sectional view of the nucleic acid extraction container.

[0055] Figure 25 for Figure 20 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0056] Figure 26 (a) and Figure 26 (b) is a diagram illustrating the nucleic acid extraction container of the fifth embodiment of the present invention.

[0057] Figure 27 for Figure 26 (a) shows the AA cross-sectional view of the nucleic acid extraction container.

[0058] Figure 28 for Figure 26 (a) shows a BB cross-sectional view of the nucleic acid extraction container.

[0059] Figure 29 for Figure 26 (a) shows a CC cross-sectional view of the nucleic acid extraction container.

[0060] Figure 30 for Figure 26 (a) shows a DD cross-sectional view of the nucleic acid extraction container.

[0061] Figure 31 for Figure 26 (a) shows an EE cross-sectional view of the nucleic acid extraction container.

[0062] Figure 32 for Figure 26 (a) is an FF cross-sectional view of the nucleic acid extraction container.

[0063] Figure 33 for Figure 26 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0064] Figure 34 (a) and Figure 34 (b) is a diagram illustrating the pressure plate of the nucleic acid extraction container according to the fifth embodiment of the present invention.

[0065] Figure 35 for Figure 34 (a) shows the JJ section view of the pressure plate.

[0066] Figure 36 for Figure 34 The HH section view of the pressure plate shown in (a).

[0067] Figure 37 for Figure 34 Figure (a) shows the GG and II sections of the pressure plate.

[0068] Figure 38 A graph showing the relationship between the volume of extract and DNA concentration when the volume of the biological material collection area is 20 μL. Detailed Implementation

[0069] The present invention will now be described based on the accompanying drawings and preferred embodiments. These embodiments are illustrative and not intended to limit the invention; all features and combinations thereof described in the embodiments are not necessarily essential to the invention.

[0070] [First Implementation Method]

[0071] Figure 1 (a) and Figure 1 (b) is a diagram illustrating the nucleic acid extraction container 10 of the first embodiment of the present invention. Figure 1 (a) is a top view of the nucleic acid extraction container 10. Figure 1 (b) is the front view of the nucleic acid extraction container 10. Figure 2 for Figure 1 (a) shows the AA cross-sectional view of the nucleic acid extraction container. Figure 3 for Figure 1 (a) shows a BB cross-sectional view of the nucleic acid extraction container. Figure 4 for Figure 1 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0072] The nucleic acid extraction container 10 is composed of a resin substrate 12, a first sealing film 24 attached to the lower surface 12a of the substrate 12, and two sealing films (a second sealing film 26 and a third sealing film 28) attached to the upper surface 12b of the substrate 12. The substrate 12 has groove-shaped first flow path 14, second flow path 16, third flow path 18 and filter section 20 with hydrophilic filter 22 formed on the lower surface 12a and upper surface 12b. The first sealing film 24 is used to seal a part of the first flow path 14 and the second flow path 16.

[0073] The substrate 12 is preferably made of a material that is stable to temperature changes and is not easily corroded by the sample solution used. Furthermore, the substrate 12 is preferably made of a material with good formability, transparency, barrier properties, and low self-fluorescence. Examples of such materials include inorganic materials such as glass, resins such as polypropylene, acrylic, polyester, and silicone, with cyclic olefin polymer resins (COP) being preferred. An example of the dimensions of the substrate 12 is a long side of 72 mm, a short side of 26 mm, and a thickness of 4 mm. For ease of handling, a long side of 50 to 200 mm is particularly preferred. From a formability perspective, a thickness of 1 to 4 mm is preferred. From a cost-effectiveness perspective, a short side of 10 to 50 mm is preferred. This substrate can be manufactured by injection molding, casting, or machining using a CNC machine tool.

[0074] Regarding the first sealing film 24 and the second sealing film 26, one of their main surfaces may be adhesive, or a functional layer that exhibits adhesiveness and adhesion through pressing, ultraviolet light irradiation, or heating may be formed on one of their main surfaces. The first sealing film 24 and the second sealing film 26 each have the function of easily and integrally adhering to the lower surface 12a and the upper surface 12b of the substrate 12. It is preferable that the first sealing film 24 and the second sealing film 26 are formed of a material containing an adhesive and having low self-fluorescence. From this perspective, transparent films made of resins such as cyclic olefin polymer resins, polyesters, polypropylene, or acrylics are suitable, but not limited to these. Furthermore, the first sealing film 24 and the second sealing film 26 can be formed of plate-shaped glass or resin. In this case, rigidity is expected, thus helping to prevent warping or deformation of the nucleic acid extraction container 10. It is preferable that the adsorption of proteins in the portions of the first sealing film 24 and the second sealing film 26 that are in contact with the flow path is minimal.

[0075] A first flow path 14, a second flow path 16, and a third flow path 18 in the form of grooves are formed on the lower surface 12a and the upper surface 12b of the substrate 12. In the nucleic acid extraction container 10 of this embodiment, most of the first flow path 14, the second flow path 16, and the third flow path 18 are formed as grooves exposed on the lower surface 12a and the upper surface 12b of the substrate 12. This is to allow for inexpensive and easy molding using injection molding with a mold or the like. To seal the grooves as flow paths, a first sealing film 24 is attached to the lower surface 12a of the substrate 12, and a second sealing film 26 is attached to the upper surface 12b. An example of the dimensions of the first flow path 14 and the second flow path 16 is a width of 1.0 mm and a depth of 1.0 mm. An example of the dimensions of the third flow path 18 is a width of 0.6 mm and a depth of 0.6 mm. From the viewpoint of reducing the overall volume of the third flow path, it is preferable that the average cross-sectional area of ​​the third flow path 18 is smaller than the average cross-sectional area of ​​the first flow path 14. Therefore, applying pressure to the first flow path 14 during filtration can reduce the amount of sample entering the third flow path 18 and prevent the extract from remaining in the third flow path 18 during the recovery of the extract.

[0076] The first flow path 14 connects the sample injection port 30 and the biomaterial collection area 20a of the filter section 20. The sample injection port 30 is formed to be exposed on the upper surface 12b of the substrate 12. The sample injection port 30 can be formed to fit well with the syringe. For example, a cylindrical tube can extend from the substrate 12. Furthermore, in order to stably fix the syringe in which the sample is inserted, the sample injection port 30 can be formed to be connected to the syringe in a Luer lock manner.

[0077] The sample is introduced into the first flow path 14 through the sample injection port 30. A pre-filter for removing foreign matter, with a pore size larger than that of the filter 22, can be installed in the first flow path 14.

[0078] A circular filter section 20 is formed and exposed on the upper surface 12b of the substrate 12. The filter section 20 is sealed by a second sealing membrane 26. A circular hydrophilic filter 22 is disposed on the filter section 20. Preferably, the filter section is formed in a way that allows for visual confirmation of the presence of a sample on the filter 22 during nucleic acid extraction; for example, a transparent membrane can be used as the second sealing membrane 26. Figure 2 As shown, the filter section 20 has a biomaterial collection area 20a for collecting biomaterial containing nucleic acids on the filter 22 and a sample passage area 20b for the sample passing through the filter 22. Here, if the filter 22 is close to a hydrophilic substance in the sample passage area 20b, water can easily remain between the filter 22 and the biomaterial when air is injected to discharge the sample from the flow path after collecting the biomaterial with the filter 22. If water remains, the nucleic acid extract can easily pass through the filter 22 during nucleic acid extraction. Therefore, it is undesirable for the filter 22 to be close to a hydrophilic substance in the sample passage area 20b.

[0079] The filter 22 is hydrophilic. Examples of materials used for this filter include polyvinylidene fluoride (PVDF), glass fiber, polyethersulfone (PES), polyester, and cellulose, with PVDF and PES being preferred as they do not readily adsorb proteins. The pore size of the filter 22 can be appropriately selected based on the biological material being collected. For example, the pore size of the filter 22 may be 1 μm or less.

[0080] Here, nucleic acids include DNA, RNA, and their derivatives. In addition, biological materials may include microorganisms such as bacteria and viruses, besides the substances that constitute living organisms.

[0081] In the biological material collection area 20a, a filter 22 is secured by an O-ring 32. An example of the filter's dimensions is a diameter of 4 mm, and an example of the O-ring's dimensions is an outer diameter of 4 mm and an inner diameter of 2 mm. Figure 2 As shown, the O-ring 32 has notches 32a and 32b to allow the sample to be fed onto the filter 22 from the first flow path 14 and the nucleic acid extract to be fed onto the filter 22 from the third flow path 18. The notch 32a on the first flow path side is formed on the upper side of the O-ring 32, but it can also be formed on the lower side. The notch 32b on the third flow path side is formed on the lower side of the O-ring 32. This is to allow for the recovery of all the nucleic acid extract on the filter 22.

[0082] The second flow path 16 connects the sample outlet 34 and the sample permeation area 20b, so that the liquid that has passed through the filter 22 passes through the sample permeation area 20b and is discharged from the sample outlet 34 through the second flow path. Figure 2 As shown, in this embodiment, the sample outlet 34 is formed to be exposed on the upper surface 12b of the substrate 12, but it may also be formed to be exposed on the lower surface 12a.

[0083] The third flow path 18 connects the air vent 36 and the biomaterial collection area 20a of the filter section 20. The air vent 36 is formed to be exposed on the upper surface 12b of the substrate 12. Figure 1As shown in (a), the third sealing membrane 28 is attached to the air vent 36. A third sealing membrane 28 with dimensions capable of sealing the air vent 36 is used. When the sample passes through the filter 22 from the sample injection port 30 via the first flow path, the air vent 36 is sealed by the third sealing membrane 28 to prevent the sample from entering the third flow path. When extracting the biological material collected on the filter 22 using nucleic acid extraction solution, the third sealing membrane 28 is peeled off, opening the air vent 36. Thus, the air vent can be opened and closed relative to the outside, thereby allowing nucleic acid extraction from the filter 22. In this embodiment, the air vent 36 is opened and closed by the third sealing membrane, but it can also be opened and closed by inserting a resin or metal plug, etc.

[0084] A pipette, for example, can be used to introduce nucleic acid extract into the biomaterial collection area 20a of the filter section 20 from either the sample injection port 30 or the air vent 36. The amount of nucleic acid extract can be enough to contact the entire surface of the filter 22, or it can contact a portion of the surface and be moved to contact the entire surface. A pipette, for example, can be used to recover the extract that has been in contact with the surface of the filter 22 from either the air vent 36 or the sample injection port 30. The air vent 36 can be configured to fit into the tip of the pipette.

[0085] like Figure 1 As shown in (a), the first flow path 14 and the third flow path 18 preferably extend toward opposite sides of each other, sandwiching the biological material collection area 20a, and more preferably the first flow path 14 and the third flow path 18, which communicate with the filter section 20, are in a straight line. This prevents sample residue from remaining in the first flow path 14 and the filter section 20 after the sample has passed through the filter 22.

[0086] In the nucleic acid extraction container 10, all flow paths are formed in a straight line, but the shape of the flow paths is not limited to this. For example, the flow paths can be formed in a so-called continuously zigzagging snake shape that combines curved and straight sections, and their width can also widen midway. Multiple filter sections 20 can also be connected in series. As a result, the area occupied by the filter sections 20 on the substrate 12 can be increased, and more biological material can be collected.

[0087] (Variation Example 1)

[0088] Next, a variation of the nucleic acid extraction container of the first embodiment will be described. Figure 5 This is a diagram illustrating a modified example 1 of a nucleic acid extraction container. Figure 5 The nucleic acid extraction container 50 shown is... Figure 1The difference in the nucleic acid extraction container 10 shown in (a) is that a nucleic acid extraction liquid injection port 52 is further provided on the upper surface 12b of the substrate 12, and a first branch flow path 54 connected to the nucleic acid extraction liquid injection port 52 is connected to a third flow path 18. In addition, the first branch flow path 54 may also be connected to the first flow path 14.

[0089] (Variation Example 2)

[0090] Figure 6 This is a diagram illustrating a modified example 2 of a nucleic acid extraction container. Figure 6 The nucleic acid extraction container 60 shown is... Figure 1 The difference in the nucleic acid extraction container 10 shown in (a) is that a nucleic acid extraction outlet 62 is further provided on the upper surface 12b of the substrate 12, and a second branch flow path 64 connected to the nucleic acid extraction outlet 62 is connected to the third flow path 18. In addition, the second branch flow path 64 can also be connected to the first flow path 14.

[0091] (Variation Example 3)

[0092] In the implementation, the above-described modified examples 1 and 2 can be combined to provide a nucleic acid extract injection port, a nucleic acid extract outlet, a first branch flow path, and a second branch flow path on the substrate 12.

[0093] (Variation Example 4)

[0094] In this implementation, nucleic acid extraction solution can be pre-sealed into either the third flow path or the first flow path, and air can be sealed between the area containing the nucleic acid extraction solution and the filter section. This simplifies the nucleic acid extraction process.

[0095] (Variation Example 5)

[0096] In this embodiment, when the sample permeation area of ​​the filter section is large, a filter with a large pore size can be used. In this case, the nucleic acid extract will pass through the filter, but by applying a small pressure from the sample outlet side, the nucleic acid extract can be prevented from passing through the filter. The pressure at this point only needs to be such that air cannot pass through a wet hydrophilic filter. A miniature blower pump (MZB1001T02) manufactured by, for example, Murata Manufacturing Co., Ltd., can be used to apply a pressure of about 1 kP from the sample outlet. Furthermore, the flow path and filter section can be formed on the substrate such that the sample passes through the filter from the lower surface of the substrate to the upper surface, while the nucleic acid extract passes through the lower side of the filter.

[0097] (Variation Example Six)

[0098] In one embodiment, a channel including a first flow path, a filter section, a second flow path, and a third flow path is formed on a substrate. Considering the cost of each channel, it is preferable to fabricate multiple channels on a single substrate.

[0099] Next, the method of using the nucleic acid extraction container 10 configured as described above will be explained. First, the sample is introduced into the first flow path 14 through the sample injection port 30, and the sample is allowed to pass through the filter section 20. The method of introducing the sample is not limited to this; for example, an appropriate amount of sample can be directly introduced from the injection port using a pipette, dropper, syringe, etc. Furthermore, after the sample is dispensed or introduced using a pipette, dropper, syringe, etc., further pressure can be applied or the sample can be aspirated from the sample discharge port 34 to move the sample to the filter section.

[0100] Figure 7 This diagram illustrates the flow of a sample through the filter section. Figure 8 As indicated by arrow A, the sample moves from the first flow path 14 to the filter section 20 and passes through the filter 22. At this time, in the biomaterial collection area 20a of the filter section 20, biomaterial containing nucleic acids is collected on the filter 22. The sample passing through the filter 22 moves to the second flow path 16. Next, air is injected through the sample injection port 30, and the sample is discharged from the first flow path 14, the filter section 20, and the second flow path 16. Throughout these steps, the air vent 36 is continuously sealed by the third sealing membrane 28.

[0101] Next, the third sealing membrane 28 is peeled off, and the nucleic acid extraction solution is injected into the third flow path through the air vent 36. Figure 8 This diagram illustrates the extraction of nucleic acids using nucleic acid extraction solution on a filter. Figure 8 As shown by arrow B1, the nucleic acid extract moves from the third flow path to the filter section 20 and comes into contact with the surface of the filter 22. Alternatively, the nucleic acid extract can be injected from the sample injection port 30 into the first flow path 14 and moved towards the filter section 20.

[0102] The pore size of filter 22 is, for example, less than 1 μm, as described above, so the nucleic acid extraction solution does not permeate through filter 22 but remains on it. The amount of nucleic acid extraction solution can be either enough to contact the entire surface of filter 22 or an amount that does not. When the amount of nucleic acid extraction solution is enough to contact the entire surface of filter 22, the solution is allowed to stand on filter 22 for, for example, 1 minute, thereby extracting nucleic acid from the biological material on filter 22. When the amount of nucleic acid extraction solution is too small to contact the entire surface of filter 22, a pipette is inserted, for example, into the air vent 36 or the sample injection port 30, and air is pushed out or drawn back using the pipette, thereby extracting nucleic acid. Figure 8 As indicated by arrow B2, the nucleic acid extract is circulated back and forth on filter 22. This circulation of the nucleic acid extract allows for the extraction of nucleic acid from the biological material collected on filter 22. Alternatively, the circulation of the nucleic acid extract using a pipette can also be achieved using a miniature blower pump or similar device. This circulation can be achieved, for example, by the method described in Japanese Patent Application Laid-Open No. 2019-180418.

[0103] In cases where the sample contains many broken cells and mitochondria and nucleic acids are released from the cells, nucleic acids collected on filter 22 can be extracted by contacting the nucleic acid extraction solution at room temperature. On the other hand, in cases where the sample is bacteria or the like, nucleic acids cannot be extracted at room temperature. In such cases, nucleic acids can be extracted by applying a temperature of approximately 95°C. For example, by placing substrate 12 on a high-temperature metal plate, the temperature of the biomaterial collection area 20a can be raised to approximately 95°C, thereby allowing nucleic acids to be extracted from samples such as bacteria.

[0104] Regarding the solution containing the extracted nucleic acid on filter 22, insert a pipette into the air vent 36 or sample injection port 30 to aspirate the solution, thereby recovering the solution from filter section 20. The extraction of nucleic acid from the sample is completed according to the above procedure.

[0105] [Second Implementation]

[0106] The nucleic acid extraction container of the second embodiment of the present invention also consists of a substrate, a sealing membrane attached to the substrate, and a filter. For structures common to the nucleic acid extraction container 10 of the first embodiment, the same reference numerals are used, and repeated descriptions are omitted where appropriate.

[0107] Figure 9 (a) and Figure 9 (b) is a diagram illustrating the nucleic acid extraction container of the second embodiment of the present invention. Figure 10 for Figure 9 (a) shows the AA cross-sectional view of the nucleic acid extraction container. Figure 11 for Figure 9 (a) shows a BB cross-sectional view of the nucleic acid extraction container. Figure 12 for Figure 9 (a) shows a CC cross-sectional view of the nucleic acid extraction container. Figure 13 for Figure 9 (a) shows a DD cross-sectional view of the nucleic acid extraction container. Figure 14 for Figure 9 (a) is a top view of the substrate of the nucleic acid extraction container shown. The nucleic acid extraction container 80 of the second embodiment has a different structure from the nucleic acid extraction container 10 of the first embodiment in terms of the filter section and the second flow path.

[0108] like Figure 9 As shown in (a), in the nucleic acid extraction container 80 of the second embodiment, a groove-shaped filter section 82 is formed and exposed on the upper surface 12b of the substrate 12. A rectangular hydrophilic filter 84 is disposed on the filter section 82. The filter section 82 has a biomaterial collection area 82a above the filter 84 and a sample permeation area 82b below the filter 84. Figure 9 As shown in (a), in the biomaterial collection area 82a, the filter 84 is fixed by providing silicone rubber 86 and 88 on both sides of the long side. An example of the dimensions of the filter 84 is a long side of 21 mm and a short side of 4.0 mm, and an example of the dimensions of the silicone rubber 86 and 88 is a long side of 21 mm, a short side of 1.5 mm, and a thickness of 0.5 mm. Therefore, the biomaterial collection area 82a becomes a flow path with a width of 1.0 mm and a depth of 0.5 mm. The sample permeation area of ​​the filter section 82 of the nucleic acid extraction container 80 in the second embodiment is larger than that of the filter section 20 of the nucleic acid extraction container 10 in the first embodiment. Therefore, in the second embodiment, the nucleic acid extraction efficiency can be further improved.

[0109] like Figure 9 As shown in (a), the exposed portion of filter 84 (the portion sandwiched between silicone rubbers 86 and 88) is aligned with the first flow path 14 and the third flow path 18. This facilitates the replacement of air in the flow path after the sample has passed through, and makes the movement and recovery of the extract easier.

[0110] like Figure 10 As shown, the sample permeation area 82b is connected to the first branch 90a, the second branch 90b, the third branch 90c, and the fourth branch 90d of the second flow path 90. The number of branch paths can be appropriately changed according to the shape of the filter 84.

[0111] The nucleic acid extraction container 80 of the second embodiment, configured as described above, is used in the same way as the nucleic acid extraction container 10 of the first embodiment described above, thereby allowing the extraction of nucleic acid from the sample.

[0112] The above-described variations 1 to 6 can also be applied to the nucleic acid extraction container 80 of the second embodiment.

[0113] [Third Implementation Method]

[0114] The nucleic acid extraction container of the third embodiment of the present invention is similar to that of the nucleic acid extraction container of the second embodiment, also consisting of a substrate, a sealing membrane attached to the substrate, and a filter. Regarding structures common to the nucleic acid extraction container 80 of the second embodiment, the same reference numerals are used, and repeated descriptions are omitted where appropriate.

[0115] Figure 15 (a) and Figure 15 (b) is a diagram illustrating the nucleic acid extraction container of the third embodiment of the present invention. Figure 16 for Figure 15 (a) shows the AA cross-sectional view of the nucleic acid extraction container. Figure 17 for Figure 15 (a) shows an EE cross-sectional view of the nucleic acid extraction container. Figure 18 for Figure 15 (a) is a top view of the substrate of the nucleic acid extraction container. The nucleic acid extraction container 100 of the third embodiment differs from the nucleic acid extraction container 80 of the second embodiment in that the nucleic acid extraction container 80 is provided with: a nucleic acid extraction liquid outlet 102 on the upper surface 12b of the substrate 12; a second branch flow path 104 connected to the nucleic acid extraction liquid outlet 102 and connected to a third flow path 18; an air injection port 106 on the upper surface 12b of the substrate 12; and a third branch flow path 108 connected to the air injection port 106 and connected to the third flow path 18. The nucleic acid extraction liquid outlet 102 and the air injection port 106 are sealed by a fourth sealing membrane 110. In the third embodiment, a predetermined amount of nucleic acid extraction liquid can be dispensed to the nucleic acid extraction liquid outlet 102 by using the region C of the third flow path 18 between the connection position of the third flow path 18 and the second branch flow path 104 and the connection position of the third flow path 18 and the third branch flow path 108.

[0116] like Figure 17 As shown, the nucleic acid extraction outlet 102 can be shaped to store the nucleic acid extraction solution. Reagents for PCR amplification can be directly mixed into the nucleic acid extraction outlet 102, which contains the stored nucleic acid extraction solution.

[0117] Next, the method of using the nucleic acid extraction container 100 configured as described above will be explained. First, in the same manner as the method of using the nucleic acid extraction container 10 of the first embodiment described above, the sample is passed through the filter section 82, the biological material containing nucleic acid is collected on the filter 84, the nucleic acid extraction solution is placed in the filter section 82, and the nucleic acid is extracted on the filter 84.

[0118] Next, the pipette is inserted into the air vent 36 and actuated, thereby moving the solution containing the extracted nucleic acid on the filter 84 toward region C, which includes the third flow path 18. During nucleic acid extraction, the third sealing membrane 28 is peeled off, opening the air vent 36. Figure 19 The diagram schematically illustrates the presence of a nucleic acid-containing solution in region C of the third flow path 18. Next, the third sealing film 28 is reattached to close the air vent 36, and the sample injection port 30 is closed using a new sealing film (not shown). Then, the fourth sealing film 110 is peeled off to open the nucleic acid extraction outlet 102 and the air injection port 106. A pipette is inserted into the air injection port 106 and air is introduced, thereby delivering only the nucleic acid-containing solution present in region C to the nucleic acid extraction outlet 102. This dispenses the nucleic acid-containing solution. The nucleic acid extraction outlet 102 has the shape described above, capable of storing the solution. Furthermore, the volume of region C is set to a volume that allows direct mixing with PCR amplification reagents, thereby storing this volume of solution in the nucleic acid extraction outlet 102. In this case, reagents for PCR amplification can be directly added to the solution stored in the nucleic acid extraction outlet 102.

[0119] The above-described variations 1, 4 to 6 can also be applied to the nucleic acid extraction container 100 of the third embodiment.

[0120] [Fourth Implementation Method]

[0121] Figure 20 (a) and Figure 20 (b) is a diagram illustrating the nucleic acid extraction container of the fourth embodiment of the present invention. Figure 21 for Figure 20 (a) shows the AA cross-sectional view of the nucleic acid extraction container. Figure 22 for Figure 20 (a) shows a BB cross-sectional view of the nucleic acid extraction container. Figure 23 for Figure 20 (a) shows a CC cross-sectional view of the nucleic acid extraction container. Figure 24 for Figure 20 (a) shows a DD cross-sectional view of the nucleic acid extraction container. Figure 25 for Figure 20 (a) is a top view of the substrate of the nucleic acid extraction container shown.

[0122] The nucleic acid extraction container 120 is composed of a resin substrate 122, a first sealing film 134 attached to the lower surface 122a of the substrate 122, and two sealing films (a second sealing film 136 and a third sealing film 138) attached to the upper surface 122b of the substrate 122. The substrate 122 has groove-shaped first flow path 124, second flow path 126, third flow path 128 and a filter section 130 with a hydrophilic filter 132 formed on the lower surface 122a and the upper surface 122b. The first sealing film 134 is used to seal the second flow path 126 and the filter section 130.

[0123] The material and size of the substrate 122 are the same as those of the substrate 12 in the first embodiment. Furthermore, similar to the substrate 12 in the first embodiment, the substrate 122 can be manufactured by injection molding, casting molding, or machining using a CNC machine tool.

[0124] The structure and material of the first sealing membrane 134 and the second sealing membrane 136 are the same as those of the first sealing membrane 24 and the second sealing membrane 26 in the first embodiment. The portions of the first sealing membrane 134 and the second sealing membrane 136 that come into contact with the flow path preferably have low protein adsorption.

[0125] A first flow path 124, a second flow path 126, and a third flow path 128 in the form of grooves are formed on the lower surface 122a and the upper surface 122b of the substrate 122. Similar to the first embodiment, in the nucleic acid extraction container 120 of the fourth embodiment, most of the first flow path 124, the second flow path 126, and the third flow path 128 are also formed as grooves exposed on the lower surface 122a and the upper surface 122b of the substrate 122. This is to allow for inexpensive and easy molding using injection molding with a mold or the like. To seal the grooves as flow paths, a first sealing film 134 is attached to the lower surface 122a of the substrate 122, and a second sealing film 136 is attached to the upper surface 12b. The dimensions of the first flow path 124, the second flow path 126, and the third flow path 128 are the same as those of the first flow path 14, the second flow path 16, and the third flow path 18 in the first embodiment.

[0126] The first flow path 124 connects the sample injection port 140 and the biomaterial collection area 130a of the filter section 130. The sample injection port 140 is formed to protrude on the upper surface 122b of the substrate 122. The shape of the sample injection port 140 is not limited to the shape shown in the figure. Similar to the sample injection port 30 in the first embodiment, the sample injection port 140 can be formed to fit well with the syringe, or it can be formed to be connected to the syringe in a Luer-locking manner.

[0127] The sample is introduced into the first flow path 124 through the sample injection port 140. A pre-filter for removing foreign matter, with a pore size larger than that of the filter 132, can be installed in the first flow path 124.

[0128] A grooved filter section 130 is formed and exposed on the lower surface 122a of the substrate 122. The filter section 130 is sealed by a first sealing membrane 134. A hydrophilic filter 132 is disposed in the filter section 130. Figure 21 As shown, the filter section 130 has a biomaterial collection area 130a for collecting biomaterial containing nucleic acids on the filter 132 and a sample permeation area 130b for the sample to pass through the filter 132. As explained in the first embodiment, in order to prevent the nucleic acid extract from passing through the filter 132 during nucleic acid extraction, it is undesirable for the filter 132 to be close to hydrophilic substances in the sample permeation area 130b.

[0129] In the sample transmission region 130b, the filter 132 is fixed by providing silicone rubber 142 and 144 on both sides of its long side. Alternatively, the filter can be bonded or fused to the groove surface of the lower surface 122a of the substrate 122. An example of the dimensions of the filter 132 is a long side of 40 mm and a short side of 4.0 mm. An example of the dimensions of the silicone rubber 142 and 144 is a long side of 40 mm, a short side of 1.5 mm, and a thickness of 0.5 mm. Therefore, the sample transmission region 130b becomes a flow path with a width of 1.0 mm and a depth of 0.5 mm. The biomaterial collection region 130a is formed as a groove on the lower surface 122a of the substrate 122, and the filter 132 is provided in the sample transmission region 130b, thus becoming a flow path. An example of the dimensions of the biomaterial collection region 130a is a width of 1.0 mm and a depth of 0.5 mm. By forming the filter section 130 on the lower surface 122a of the substrate 122 and fixing the filter 132 from the sample passage area 130b side, even if foreign matter adheres to the clamp of the filter such as the silicone rubber in this embodiment, or if foreign matter enters during the assembly of the nucleic acid extraction container, these foreign matter can be prevented from mixing into the nucleic acid extraction solution. Furthermore, when the sample passes through the filter, pressure is applied to the biomaterial collection area in the substrate. Therefore, depending on the magnitude of the pressure, there is a risk that the membrane sealing the filter section may peel off. However, according to the structure of the filter section 130 in this embodiment, the area sealing the biomaterial collection area 130a is smaller, thus reducing the risk of the membrane sealing the filter section peeling off.

[0130] The material and pore size of filter 132 are the same as those of filter 22 in the first embodiment.

[0131] The second flow path 126 connects the sample outlet 146 and the sample permeation area 130b, so that the liquid that has passed through the filter 132 passes through the sample permeation area 130b and is discharged from the sample outlet 146 through the second flow path 126. In this embodiment, the sample outlet 146 is formed to protrude from the upper surface 122b of the substrate 122, but it may also be formed on the lower surface 122a.

[0132] The third flow path 128 connects the air vent 148 and the biomaterial collection area 130a of the filter section 130. The air vent 148 is formed to protrude from the upper surface 122b of the substrate 122. Figure 20 As shown in (a), the third sealing film 138 is attached to the air vent 148. The structure of the air vent 148 and the third sealing film 138 is the same as that of the air vent 36 and the third sealing film 28 in the first embodiment.

[0133] Similar to the first embodiment, nucleic acid extract can be introduced into the biomaterial collection area 130a of the filter section 130 from either the sample injection port 140 or the air vent 148. The amount of nucleic acid extract can be enough to contact the entire surface of the filter 132, or it can be enough to contact a portion of the surface and then move the extract to contact the entire surface. Similar to the first embodiment, the extract that has contacted the surface of the filter 132 can be recovered from either the air vent 148 or the sample injection port 140, or pressure can be applied to the biomaterial collection area 130a to allow the extract to pass through the filter 132 and be recovered from the sample discharge port 146. The air vent 148 can be configured to fit into the tip of a pipette.

[0134] like Figure 20 As shown in (a), the first flow path 124 and the third flow path 128 preferably extend toward opposite sides, sandwiching the biological material collection area 130a, and more preferably, the first flow path 124 and the third flow path 128, which communicate with the filter section 130, are in a straight line. This prevents sample residue from remaining in the first flow path 124 and the filter section 130 after the sample has passed through the filter 132.

[0135] In the nucleic acid extraction container 120, all flow paths are formed in a straight line, but the shape of the flow paths is not limited to this. For example, the flow paths can be formed in a so-called continuously zigzagging snake shape that combines curved and straight sections, and their width can also widen midway. Multiple filter sections 130 can also be connected in series. As a result, the area occupied by the filter sections 130 on the substrate 122 can be increased, and more biological material can be collected.

[0136] The nucleic acid extraction container 120 of the fourth embodiment, configured as described above, is used in the same way as the nucleic acid extraction container 10 of the first embodiment described above, thereby allowing the extraction of nucleic acid from the sample.

[0137] The above-described variations 1 to 6 can also be applied to the nucleic acid extraction container 120 of the fourth embodiment.

[0138] [Fifth Implementation]

[0139] Figure 26 (a) and Figure 26 (b) is a diagram illustrating the nucleic acid extraction container of the fifth embodiment of the present invention. Figure 27 for Figure 26 (a) shows the AA cross-sectional view of the nucleic acid extraction container. Figure 28 for Figure 26 (a) shows a BB cross-sectional view of the nucleic acid extraction container. Figure 29 for Figure 26 (a) shows a CC cross-sectional view of the nucleic acid extraction container. Figure 30 for Figure 26 (a) shows a DD cross-sectional view of the nucleic acid extraction container. Figure 31 for Figure 26 The EE cross-sectional view of the nucleic acid extraction container is shown in (a). Figure 32 for Figure 26 (a) is an FF cross-sectional view of the nucleic acid extraction container. Figure 33 for Figure 26 (a) is a top view of the substrate of the nucleic acid extraction container shown. Figure 34 (a) and Figure 34 (b) is a diagram illustrating the pressure plate of the nucleic acid extraction container according to the fifth embodiment of the present invention.

[0140] Figure 35 for Figure 34 (a) shows the JJ section view of the pressure plate. Figure 36 for Figure 34 The HH section view of the pressure plate shown in (a). Figure 37 for Figure 34 Figure (a) shows the GG and II sections of the pressure plate.

[0141] The nucleic acid extraction container 160 is composed of a resin substrate 162, a first sealing film 170, and a pressure plate 180. The substrate 162 has grooved flow paths 164 and a filter section 166 with a hydrophilic filter 168 formed on its lower surface 162a and upper surface 162b. The first sealing film 170 is attached to the lower surface 162a of the substrate 162 to seal the flow paths 164 and the filter section 166. The pressure plate 180 fixes the filter 168 to the filter section 166.

[0142] The substrate 162 is made of the same material and has the same dimensions as the substrate 12 in the first embodiment. Furthermore, similar to the substrate 12 in the first embodiment, the substrate 162 can be manufactured by injection molding, casting, or machining using a CNC machine tool.

[0143] The structure and material of the first sealing membrane 170 are the same as those of the first sealing membrane 24 and the second sealing membrane 26 in the first embodiment. The portion of the first sealing membrane 170 that comes into contact with the flow path preferably has low protein adsorption.

[0144] A groove-shaped flow path 164 is formed on the lower surface 162a of the substrate 162. In the nucleic acid extraction container 160 of the fifth embodiment, the flow path 164 is formed as a groove exposed on the lower surface 162a of the substrate 162. This is to allow for inexpensive and easy molding using injection molding with a mold or the like. To seal the groove as a flow path, a first sealing film 170 is attached to the lower surface 162a of the substrate 162. The dimensions of the flow path 164 are the same as those of the second flow path 16 in the first embodiment.

[0145] The sample injection port 174 is connected to the biomaterial collection area 130a of the filter section 166. The sample injection port 174 is formed to protrude from the upper surface 162b of the substrate 162. The shape of the sample injection port 174 is not limited to the shape shown in the figure. Similar to the sample injection port 30 in the first embodiment, the sample injection port 174 can be formed to fit well with the syringe, and the sample injection port 140 can be formed to be connected to the syringe in a Luer locking manner.

[0146] The sample is introduced from the sample injection port 174 into the biomaterial collection area 166a of the filter section 166. A pre-filter for removing foreign matter, with a pore size larger than that of the filter 168, can be installed in the sample injection port 174.

[0147] A grooved filter section 166 is formed and exposed on the lower surface 162a of the substrate 162. A hydrophilic filter 168 is provided in the filter section 166. Figure 27As shown, the filter section 166 has a biomaterial collection region 166a for collecting biomaterial containing nucleic acids on the filter 168 and a sample passage region 166b for the sample passing through the filter 168. In the sample passage region 166b, the filter 168 is fixed by a pressure plate 180, but the filter 168 can also be bonded or welded to the groove surface of the lower surface 162a of the substrate 162. The filter section 166 is sealed by a first sealing membrane 170. As described in the first embodiment, in order to prevent nucleic acid extract from passing through the filter 168 during nucleic acid extraction, it is undesirable for the filter 168 to be near hydrophilic substances in the sample passage region 166b.

[0148] An example of the dimensions of filter 168 is 46 mm on the long side and 6 mm on the short side, and an example of the dimensions of pressure plate 180 is 46 mm on the long side, 6 mm on the short side, and 1 mm thick. The sample passage area 166b becomes a flow path with a width of 1 mm and a depth of 1 mm. The biomaterial collection area 166a is formed as a groove on the lower surface 162a of substrate 162, and filter 168 is provided in sample passage area 166b, thereby becoming a flow path. An example of the dimensions of biomaterial collection area 166a is 1 mm wide and 0.5 mm deep. By forming filter portion 166 on the lower surface 162a of substrate 162 in this way, and fixing filter 168 from the sample passage area 166b side by pressure plate 180, even if foreign matter adheres to the pressure plate in this embodiment or foreign matter enters during the assembly of nucleic acid extraction container, these foreign matter can be prevented from mixing into the nucleic acid extraction solution. Furthermore, when the sample is passed through the filter, pressure is applied to the biomaterial collection area in the substrate. Therefore, depending on the magnitude of the pressure, there is a risk that the membrane sealing the filter section may be peeled off. However, according to the structure of the filter section 166 in this embodiment, the area of ​​the biomaterial collection area 166a is reduced, and the risk of the membrane sealing the filter section being peeled off is reduced.

[0149] The material and pore size of filter 168 are the same as those of filter 22 in the first embodiment.

[0150] like Figure 34 of (a), Figure 34 (b) Figures 35-37 As shown, the pressure plate 180 is made of a resin substrate 182, and a notch 182a constituting the sample transmission area 166b is formed on the substrate 182. The material of the substrate 182 is the same as that of the substrate 12 in the first embodiment. The size and shape of the substrate 182 can be appropriately adjusted according to the desired size and shape of the filter section 166. The size and shape of the notch 182a can be appropriately adjusted according to the desired size and shape of the sample transmission area 166b. Furthermore, similar to the substrate 12 in the first embodiment, the substrate 182 can be manufactured by injection molding, casting molding, or machining using a CNC machine tool.

[0151] The flow path 164 connects the sample outlet 176 and the sample permeation area 166b, so that the liquid that has passed through the filter 168 passes through the sample permeation area 166b and is discharged from the sample outlet 176 through the flow path 164. In this embodiment, the sample outlet 176 is formed to protrude from the upper surface 162b of the substrate 162, but it may also be formed on the lower surface 162a.

[0152] The air vent 178 is connected to the biomaterial collection area 166a of the filter section 166. The air vent 178 is formed to protrude from the upper surface 166b of the substrate 162. Figure 26 As shown in (a), the second sealing film 172 is attached to the air vent 178. The structure of the air vent 178 and the second sealing film 172 is the same as that of the air vent 36 and the third sealing film 28 in the first embodiment.

[0153] Similar to the first embodiment, nucleic acid extract can be introduced into the biomaterial collection area 166a of the filter section 166 from either the sample injection port 174 or the air vent 178. The amount of nucleic acid extract can be enough to contact the entire surface of the filter 168, or it can be enough to contact a portion of the surface and then move the extract to contact the entire surface. Similar to the first embodiment, the extract that has contacted the surface of the filter 168 can be recovered from either the air vent 178 or the sample injection port 174, or pressure can be applied to the biomaterial collection area 166a to allow the extract to pass through the filter 168 and be recovered from the sample discharge port 176. The air vent 178 can be configured to fit into the tip of a pipette.

[0154] like Figure 26 (a) and Figure 27 As shown, the sample injection port 174 and the air communication port 178 are directly connected to the biomaterial collection area 166a. Furthermore, the number of sealing films is less than that of the first to fourth embodiments. Therefore, the nucleic acid extraction container of the fifth embodiment can be manufactured at a lower cost than that of the first to fourth embodiments.

[0155] like Figure 26 (a) and Figure 27 As shown, the sample injection port 174 and the air communication port 178 are preferably arranged on opposite sides of each other, sandwiching the biomaterial collection area 166a. This prevents sample residue from remaining in the filter section 166 after the sample has passed through the filter 168.

[0156] In the nucleic acid extraction container 160, the flow path is formed in a straight line, but the shape of the flow path is not limited to this. For example, the flow path can be formed into a so-called continuously zigzagging snake shape that combines curved and straight sections, and its width can also widen midway. Multiple filter sections 166 can also be connected in series. As a result, the area occupied by the filter section 166 on the substrate 162 can be increased, and more biological material can be collected.

[0157] The nucleic acid extraction container 160 of the fifth embodiment, configured as described above, is used in the same way as the nucleic acid extraction container 10 of the first embodiment described above, thereby allowing the extraction of nucleic acid from the sample.

[0158] The above-described variations 5 and 6 can also be applied to the nucleic acid extraction container 160 of the fifth embodiment.

[0159] (Example)

[0160] The following describes embodiments of the present invention, but these embodiments are merely examples for illustrating the present invention in a preferred manner and do not limit the present invention in any way.

[0161] In this embodiment, the analysis of environmental DNA compares the method using nucleic acid extraction solutions implemented in this embodiment with the existing standard method. Here, environmental DNA (eDNA) refers to DNA released into the environment from organisms living there. More specifically, various organisms living in the environment continuously release their DNA into their surroundings. For example, in the case of animals, DNA is released through skin, body hair, excrement, carcasses, etc. By detecting or quantifying environmental DNA present in the environment, the species living in that environment can be identified, or the biodiversity of that environment can be assessed. Furthermore, from a stability perspective, the type of DNA analyzed is mostly mitochondrial DNA. In the standard method, several hundred mL to 1 L of sample water is filtered, and DNA is extracted from the collected particles containing environmental DNA, ultimately yielding 200 μL of liquid. A portion of this liquid (approximately 2 μL) is then used for PCR detection to observe the presence or extent of the organism being tested.

[0162] The reagents used in the PCR in this embodiment are shown in Table 1 below.

[0163] [Table 1]

[0164]

[0165]

[0166] The sequences of the forward primer (primer F), reverse primer (primer R), and sampling needle used in PCR are as follows.

[0167] Rainbow trout primer F: 5'-AGTCTCTCCCTGTATATCGTC-3' (serial number 1)

[0168] Rainbow trout primer R: 5'-GATTTAGTTCATGAAGTTGCGAGAGTA-3' (serial number 2)

[0169] Rainbow trout sampling needle: 5'-CCAACAACTCTTTAACCATC-3' (Serial No. 3)

[0170] The 5' end of the sampling needle is marked with FAM, and the 3' end is marked with [NFQ]-[MGB].

[0171] (Reference: Wilcox, TM, Carim, KJ, McKelvey, KS, Young, MK, & Schwartz, MK2015 (4Nov.). The dual challenges of generality and specificity when developing environmental DNAmarkers for species and subspecies of Oncorhynchus.PLoS ONE,10(11)e0142008.doi:10.1371 / journal.pone.0142008.)

[0172] Carp primer F: 5'-GGTGGGTTCTCAGTAGACAATGC-3' (serial number 4)

[0173] Carp primer R: 5'-GGCGGCAATAACAAATGGTAGT-3' (serial number 5)

[0174] Carp sampling needle: 5'-CACTAACACG ATTCTTCGCA TTCCACTTCC-3"' (Serial No. 6)

[0175] The 5' end of the sampling needle is marked with FAM, and the 3' end is marked with TAMRA.

[0176] (Reference: Takahara, T., Minamoto, T., Yamanaka, H., Doi, H. & Kawabata, Z. 2012. Estimation of fish biomass using environmental DNA. PLoS ONE, 7: e35868.)

[0177] Skipjack tuna primer F: 5'-TACCCCTGACGTAGAATCAGCC-3' (serial number 7)

[0178] Skipjack tuna primer R: 5'-GGCCAATATGGGAGTAAATGCAG-3' (serial number 8)

[0179] Skipjack tuna sampling needle; 5'-TGCCGAGACGTAAACTTCGG-3' (Serial No. 9)

[0180] The 5' end of the sampling needle is marked with Cy5, and the 3' end is marked with BHQ3.

[0181] (Reference: Lin, W.-F. & Hwang, D.-F. 2008. Application of species-specific PCR for the identification of dried bonito product (Katsuobushi). FoodChemistry 106:390-396.)

[0182] The conditions for PCR amplification are as follows.

[0183] Repeat at 95°C for 15 seconds, then repeat at 60°C for 10 seconds and at 95°C for 3.5 seconds.

[0184] (Example 1)

[0185] Production Figure 1 (a) and Figure 1 The nucleic acid extraction container 10 is shown in (b). The nucleic acid extraction container 10 is a plate-shaped container measuring 26*75*4mm, made of cyclic olefin polymer (COP). The flow paths, filter section, and air vent are sealed by attaching 3M polyolefin micro-sealing tape (9795). The first flow path 14 and the second flow path 16 are 1.0mm wide and 1.0mm deep, and the third flow path 18 is 0.6mm wide and 0.6mm deep. The hydrophilic filter has a diameter of φ4mm. The filter 22 is fixed by O-rings 32 with a diameter of φ4*φ2. The filter 22 is made of polyvinylidene fluoride (PVDF) with a pore size of 0.45μm. The effective surface area of ​​the filter 22 is φ3mm.

[0186] Using this nucleic acid extraction container, the following steps are taken to obtain an extract from which DNA has been extracted.

[0187] (1) Insert a 10mL syringe into the sample injection port 30 and inject 2mL of sample from there. Then, insert the tube into the sample discharge port 34 and discard the discharged solution.

[0188] (2) Temporarily remove the previous syringe, draw in 10 mL of air, and then insert it again into the sample injection port 30 to inject 10 mL of air into the flow path, thereby discharging the sample in the first flow path 14 and the second flow path 16.

[0189] (3) Peel off the sealing film of the air vent 36 and inject 5 μL of DNA extraction solution through a pipette. While visually confirming the position, push the DNA extraction solution into the filter section 20 and let it stand for 1 minute.

[0190] (4) Insert the pipette into the air vent 36 to aspirate the previously extracted liquid.

[0191] The sample was diluted with water from a suspension obtained by suspending raw bonito flesh in pure water. A 200 mM Na₂CO₃ aqueous solution was used as the DNA extraction solution.

[0192] 1.6 μL of the extract obtained using nucleic acid extraction container 10 was mixed with 14.4 μL of PCR amplification reagent and amplified using a PCR1100 manufactured by Nippon Sheet Glass Co., Ltd., resulting in a cycle number threshold (Ct value) of 38.9. The time required for filtration and extraction was approximately 3 minutes. In this embodiment, the extract and PCR amplification reagent were mixed directly, but it is also possible to mix the extract with a neutralizing solution before mixing it with the PCR reagent.

[0193] (Comparative Example 1)

[0194] Following the "Environmental DNA Survey and Experiment Manual (ver 2.1)" (General Incorporated Association for Environmental DNA, Internet <URL: http: / / ednasociety.org / eDNA_manual_ver2_1_3.pdf>), after filtering 50 mL of the same sample as in Example 1 through a cartridge-type filter (Sterivex 0.45 μm; manufactured by Merck), DNA was extracted using the QIAGEN KK DNeasy Blood and Tissue Kit. 1.6 μL of the extracted solution was mixed with 14.4 μL of PCR amplification reagent and amplified using the same mobile PCR device as in Example 1 (product name PicoGenePCR1100), yielding a Ct value of 40.1. The filtration and extraction process took approximately 2 hours.

[0195] The results of Example 1 and Comparative Example 1 are shown in Table 2 below.

[0196] [Table 2]

[0197]

[0198] The amount of sample in Example 1 was less than 1 / 10 of that in Comparative Example 1. Furthermore, the process from filtration to extraction in Example 1 was simpler than in Comparative Example 1, and the process time in Example 1 was approximately 3 minutes, while in Comparative Example 1 it took 2 hours. Further, as shown in Table 2, the Ct value obtained through Example 1 was nearly equivalent to the Ct value obtained through Comparative Example 1, indicating that Example 1 can achieve the same detection sensitivity as Comparative Example 1 with a sample amount of only 1 / 10. Example 1 used fewer consumables, and the nucleic acid extraction container 10 used for extraction had a simple structure, allowing for low-cost manufacturing. Typically, in nucleic acid testing, many items are disposable to avoid contamination such as residues, making it crucial to reduce the cost of consumables. Furthermore, the small sample amount in Example 1 facilitated the delivery of the water sample to the testing company. Additionally, Example 1 required no special equipment and had a few simple steps, allowing anyone to easily perform extraction, even on-site. Moreover, in Example 1, filtration and extraction could be performed in a sealed space, thus reducing the risk of contamination.

[0199] (Example 2)

[0200] Production Figure 9 (a) and Figure 9 The nucleic acid extraction container 80 is shown in (b). The nucleic acid extraction container 80 is a plate-shaped container measuring 26*75*4mm, made of cyclic olefin polymer (COP). The flow paths, filter section, and air vent are sealed by attaching 3M polyolefin micro-sealing tape (9795). The first flow path 14 and the second flow path 16 are 1.0mm wide and 1.0mm deep, and the third flow path 18 is 0.6mm wide and 0.6mm deep. The filter 84 is 21mm long and 4mm wide. The filter 84 is fixed by pressing down on both sides with silicone rubber 86 and 88, each 1.5mm wide and 0.5mm thick. The filter 84 is made of polyvinylidene fluoride (PVDF) with a pore size of 0.45μm. The effective area of ​​the filter 84 is 20mm². 2 .

[0201] Using the nucleic acid extraction container 80, the following steps are taken to obtain the DNA extract.

[0202] (1) Insert a 10mL syringe into the sample injection port 30 and inject 10mL or 5mL of sample from here. At this time, insert the tube into the sample discharge port 34 and discard the discharged solution.

[0203] (2) Temporarily remove the previous syringe, draw in 10 mL of air, and then insert it back into the sample injection port 30 to inject 10 mL of air into the first flow path and the second flow path.

[0204] (3) Peel off the third sealing membrane 28 of the air vent 36 and inject 5 μL of DNA extraction solution through a pipette, pushing the extraction solution into the filter section 82. Then, while visually confirming the position, use a pipette to circulate the DNA extraction solution back and forth for 1 minute to ensure that the DNA extraction solution contacts all effective surfaces of the filter 84.

[0205] (4) Insert the pipette into the air vent 36 to aspirate the previously extracted liquid.

[0206] The samples were 10 mL of water from a rainbow trout tank diluted 100 times with pure water (Example 2-1) and 5 mL of water from a pond in Tsukuba City where carp live (Example 2-2). For DNA extraction, a 200 mM Na₂CO₃ aqueous solution was used (Example 2-1), and the simplified extraction kit, version 2, from Kanekachi Chemical Co., Ltd. was used (Example 2-2).

[0207] Take 1.6 μL of the obtained DNA extract and mix it directly with 14.4 μL of PCR amplification reagent. Amplify the DNA using the same mobile PCR apparatus as in Example 1, and obtain the results shown in Table 3 below. This time, the extract was mixed directly with the PCR amplification reagent, but it is also possible to mix the extract with a neutralizing solution first, etc., before mixing it with the PCR reagent. The time required for filtration and extraction is approximately 3 minutes.

[0208] [Table 3]

[0209]

[0210] In the DNA extraction of this embodiment, the DNA extraction solution only contacts about half of the filter. However, it is known that by repeatedly circulating the DNA extraction solution, DNA trapped across the entire area of ​​the filter can be extracted. Accordingly, the sensitivity can be further improved by increasing the length of the filter section and increasing the sample permeation.

[0211] Typically, nucleic acids are extracted by immersing the extract (such as a mouse tail or plant material) in an extraction solution to dissolve it. In this embodiment, it is demonstrated that extraction can be performed even without immersion in the extraction solution, simply by allowing the extraction solution to flow over a filter containing the biological material. Extraction via flow alone means that the amount of extraction solution is less than with immersion, resulting in a higher concentration of nucleic acids in the extraction solution.

[0212] (Comparative Example 2)

[0213] Similar to Comparative Example 1, DNA was extracted from 100 mL of the same sample as in Example 2-1 (Comparative Example 2-1) and from 50 mL of the same sample as in Example 2-2 (Comparative Example 2-2). 1.6 μL of the resulting extract was mixed with 14.4 μL of PCR amplification reagent and amplified using the same mobile PCR apparatus as in Example 1, yielding results as shown in Table 4 below. The time required for filtration and extraction was approximately 2 hours.

[0214] [Table 4]

[0215]

[0216] In Example 2, even using the same sample as Comparative Example 2, nearly the same Ct value was obtained with 1 / 10 of the sample amount, and the same amount of DNA was detected. That is, the detection sensitivity of Example 2 is equivalent to that of Comparative Example 2. Similar to Example 1, Example 2 has the advantages of easy sample delivery to testing companies, simple extraction steps, and less contamination.

[0217] (Example 3)

[0218] Production Figure 15 (a) and Figure 15 The nucleic acid extraction container 100 is shown in (b). The nucleic acid extraction container 100 is a plate-shaped container measuring 26*75*4mm, made of cyclic olefin polymer (COP). The flow paths, filter section, and injection port are sealed by attaching 3M polyolefin micro-sealing tape (9795). The first flow path 14 and the second flow path 16 are 1.0mm wide and 1.0mm deep. The third flow path 18 is 0.6mm wide and 0.6mm deep. The second branch flow path 104 and the third branch flow path 108 have the same dimensions as the third flow path 18. The filter 84 is 21mm long and 4mm wide. The filter 84 is fixed by pressing down on both sides with silicone rubber 86 and 88, which are 1.5mm wide and 0.5mm thick. The filter 84 is made of polyvinylidene fluoride (PVDF) with a pore size of 0.45μm. The effective area of ​​the filter 84 is 20mm². 2 .

[0219] Using the nucleic acid extraction container 100, the following steps were taken to obtain the DNA extraction solution. The sample consisted of 10 mL of water from Example 2-1.

[0220] (1) Insert a 10 mL syringe into the sample injection port 30 and inject 10 mL of sample from here. At this time, insert the tube into the discharge port and discard the discharged solution.

[0221] (2) Temporarily remove the previous syringe, draw in 10 mL of air, and then reinsert it into the sample injection port 30 to inject 10 mL of air into the flow path.

[0222] (3) Peel off the third sealing membrane 28 and inject 5 μL of extract through the air vent 36 using a pipette. After pushing the extract into the filter 84, while visually confirming the position, use a pipette to make the extract circulate back and forth for 1 minute so that the extract comes into contact with all effective surfaces of the filter 84, and finally stop the extract in the area C including the third flow path 18.

[0223] (4) After sealing the air inlet 36 with the third sealing film and sealing the sample injection port 30 with the new sealing film, peel off the fourth sealing film 110, insert the pipette into the air injection port 106, and push air in through the pipette, thereby storing the previous extract in the liquid reservoir of the nucleic acid extract outlet 102.

[0224] The extract stored in the reservoir was dispensed as a 1.6 μL aliquot. This was then mixed with 14.4 μL of PCR amplification reagent. The DNA in the mixture was amplified using the same mobile PCR apparatus as in Example 1, yielding a Ct value of 30.2. The time required for filtration and extraction was approximately 3 minutes. In this example, the extract was mixed directly with the PCR amplification reagent, but it is also possible to mix the extract with a neutralizing solution before mixing it with the PCR reagent. Compared to Example 2, the 1.6 μL aliquot is not required, and the tube for mixing with the PCR amplification reagent is no longer needed, thus making the transition from extraction to PCR amplification easier.

[0225] [Table 5]

[0226]

[0227] (Example 4)

[0228] Production Figure 26 (a) and Figure 26 The nucleic acid extraction container 160 is shown in (b). The nucleic acid extraction container 160 is a plate-shaped container measuring 26*75*4mm, made of cyclic olefin polymer (COP). The flow path, filter section, and injection port are sealed by attaching 3M polyolefin micro-sealing tape (9795). The flow path 164 is 1.0mm wide and 1.0mm deep. The sample permeation area 166b is a flow path 1mm wide and 1mm deep. The biomaterial collection area 166a is a flow path 1mm wide and 0.5mm deep. The filter 168 is 46mm long and 6mm wide. The flow path is sealed by attaching 3M polyolefin micro-sealing tape (9795). Figure 34 The pressure plate 180 is placed inside the nucleic acid extraction container 160 to fix the filter 168. The pressure plate 180 is a plate with dimensions of 6*46*1mm. The filter 168 is made of polyvinylidene fluoride (PVDF) with a pore size of 0.65μm. The effective area of ​​the filter 168 is 40mm². 2The volume of the biomaterial collection area 166a is 20 μL.

[0229] Using the nucleic acid extraction container 160, the following steps are taken to obtain an extract from which DNA was extracted.

[0230] (1) Insert a 10 mL syringe into the sample injection port 174 and inject 10 mL of sample from there. Then, insert the tube into the discharge port and discard the discharged solution.

[0231] (2) Temporarily remove the previous syringe, draw in 10 mL of air, and then reinsert it into the sample injection port 174 to inject 10 mL of air into the flow path.

[0232] (3) Peel off the second sealing film 172, inject the specified amount of extract through the air vent 178 using a pipette, push the extract into the filter 168, and while visually confirming the position, use a pipette to make the extract back and forth for 2 minutes so that the extract comes into contact with all effective surfaces of the filter 168.

[0233] (4) Use the same pipette to recover the extract from the air vent 178.

[0234] The sample consisted of 10 mL of water from the rainbow trout tank diluted 50 times with pure water. The DNA extraction solution was prepared using the simplified extraction kit, version 2, from Kaneyuki Chemical Industries, Ltd.

[0235] Take 1.5 μL of the obtained extract and mix it directly with 13.5 μL of PCR amplification reagent. Amplification is then performed using the StepOne Plus real-time PCR system (Applied Biosystems). The relationship between extract volume and DNA concentration (replication number / μL) is as follows: Figure 38 As shown in the figure. The DNA concentration is derived from the Ct value used to create the standard curve during PCR.

[0236] according to Figure 38 It can be seen that if a small amount of extraction solution is used for extraction, the DNA concentration of the extraction solution will be higher.

[0237] The present invention has been described above based on embodiments. Those skilled in the art will understand that this embodiment is an example, and various modifications can be produced by combining the components and processes of these embodiments, all of which fall within the scope of the present invention.

[0238] (Industry availability)

[0239] This invention can be applied to nucleic acid extraction containers and nucleic acid extraction methods.

[0240] (Explanation of reference numerals in the attached diagram)

[0241] 10: Nucleic acid extraction container; 14: First flow path; 16: Second flow path; 18: Third flow path;

[0242] 20: Filter section; 20a: Biomaterial collection area; 20b: Sample permeation area;

[0243] 22: Filter; 30: Sample injection port; 34: Sample discharge port; 36: Air vent;

[0244] 50: Nucleic acid extraction container; 52: Nucleic acid extraction solution injection port; 54: First branching flow path;

[0245] 60: Nucleic acid extraction container; 62: Nucleic acid extract outlet; 64: Second branching path;

[0246] 80: Nucleic acid extraction container; 82: Filter section; 82a: Biomaterial collection area;

[0247] 82b: Sample permeation area; 84: Filter; 90: Second flow path;

[0248] 100: Nucleic acid extraction container; 102: Nucleic acid extraction solution outlet;

[0249] 104: Second branch flow path; 106: Air injection port; 108: Third branch flow path;

[0250] 120: Nucleic acid extraction container; 124: First flow path; 126: Second flow path;

[0251] 128: Third flow path; 130: Filter section; 130a: Biomaterial collection area;

[0252] 130b: Sample transmission area; 132: Filter; 140: Sample injection port;

[0253] 146: Sample discharge port; 148: Air vent; 160: Nucleic acid extraction container;

[0254] 164: Flow path; 166: Filter section; 166a: Biomaterial collection area;

[0255] 166b: Sample transmission area; 168: Filter; 174: Sample injection port;

[0256] 176: Sample outlet; 178: Air connection port.

[0257] (Plain text sequence list)

[0258] Serial No. 1: Rainbow trout forward PCR primers

[0259] Serial No. 2: Rainbow trout reverse PCR primers

[0260] Serial Number 3: Rainbow Trout Sampling Needle

[0261] Serial number 4: Carp forward PCR primers

[0262] Serial number 5: Carp reverse PCR primers

[0263] Serial Number 6: Carp Sampling Needle

[0264] Serial number 7: Skipjack tuna forward PCR primers

[0265] Serial No. 8: Skipjack tuna reverse PCR primers

[0266] Serial number 9: Skipjack tuna sampling needle. sequence list <110> Opto-Tech Group Japan Branch Fukuzawa Takashi <120> Nucleic acid extraction containers and nucleic acid extraction methods <130> Z285-70001WO <150> JP 2020-134388 <151> 2020-8-7 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Rainbow trout forward PCR primers <400> 1 agtctctccc tgtatatcgt c 21 <210> 2 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Rainbow trout reverse PCR primers <400> 2 gatttagttc atgaagttgc gagagta 27 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Rainbow trout sampling needle <400> 3 ccaacaactc tttaaccatc 20 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Forward PCR primers for carp <400> 4 ggtgggttct cagtagacaa tgc 23 <210> 5 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> carp reverse PCR primers <400> 5 ggcggcaata acaaatggta gt 22 <210> 6 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Carp sampling needle <400> 6 cactaacacg attcttcgca ttccacttcc 30 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Skipjack tuna forward PCR primers <400> 7 tacccctgac gtagaatcag cc 22 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Skipjack tuna reverse PCR primers <400> 8 ggccaatatg ggagtaaatg cag 23 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Skipjack tuna sampling needle <400> 9 tgccgagacg taaacttcgg 20

Claims

1. A nucleic acid extraction container, characterized by, has: a filter section including a hydrophilic filter, a biological material collection region, and a sample permeation region, wherein the filter collects biological material containing nucleic acid from a sample, the biological material collection region collects the biological material on the filter, and the sample permeation region allows passage of liquid that has permeated the filter; a sample injection port that communicates with the biological material collection region; an air communication port that communicates with the biological material collection region; and a sample discharge port that communicates with the sample permeation region, the air communication port is configured to be opened and closed with respect to the outside, the sample moves to the biological material collection region along a flow path that communicates with the biological material collection region, the liquid is discharged by moving along a flow path that communicates with the sample permeation region, and a solution containing the nucleic acid is recovered by moving along a flow path that communicates with the biological material collection region.

2. The nucleic acid extraction container according to claim 1, wherein the filter extends along the flow path of the biological material collection region.

3. The nucleic acid extraction container according to claim 1 or 2, wherein the sample injection port and the air communication port are disposed on opposite sides of each other with the biological material collection region interposed therebetween.

4. The nucleic acid extraction container according to claim 1 or 2, wherein the nucleic acid extraction container further has: a first flow path that communicates the sample injection port and the biological material collection region; a second flow path that communicates the sample discharge port and the sample permeation region; and a third flow path that communicates the air communication port and the biological material collection region.

5. The nucleic acid extraction container according to claim 4, wherein the first flow path and the third flow path extend toward opposite sides of each other with the biological material collection region interposed therebetween.

6. The nucleic acid extraction container according to claim 1 or 2, wherein the air communication port is a nucleic acid extraction solution injection port.

7. The nucleic acid extraction container according to claim 4, wherein the nucleic acid extraction container further has: a nucleic acid extraction solution injection port; and a first diverging flow path that communicates with the nucleic acid extraction solution injection port, the first diverging flow path is connected to the third flow path.

8. The nucleic acid extraction container according to claim 4, wherein the nucleic acid extraction container further has: a nucleic acid extraction solution discharge port; and a second diverging flow path that communicates with the nucleic acid extraction solution discharge port, the second diverging flow path is connected to the third flow path.

9. The nucleic acid extraction container according to claim 8, wherein the nucleic acid extraction container further has: an air injection port; and a third diverging flow path that communicates with the air injection port, the third diverging flow path is connected to the third flow path.

10. The nucleic acid extraction container according to claim 9, wherein the nucleic acid extraction container is configured such that a region of the third flow path between a connection position of the third flow path to the second diverging flow path and a connection position of the third flow path to the third diverging flow path divides a prescribed amount of extraction solution.

11. The nucleic acid extraction container according to claim 4, wherein ​ A nucleic acid extraction solution is enclosed in the third flow path, and air is enclosed between the region where the nucleic acid extraction solution is present and the filter section.

12. The nucleic acid extraction container according to claim 7, wherein A nucleic acid extraction solution is enclosed in the first branched flow path, and air is enclosed between the region where the nucleic acid extraction solution is present and the filter section.

13. The nucleic acid extraction container according to claim 9, wherein A nucleic acid extraction solution is enclosed in the third branched flow path, and air is enclosed between the region where the nucleic acid extraction solution is present and the filter section.

14. The nucleic acid extraction container according to claim 4, wherein The average cross-sectional area of the third flow path is smaller than the average cross-sectional area of the first flow path.

15. The nucleic acid extraction container according to claim 1 or 2, wherein The filter section is configured so that it is possible to visually confirm from the outside whether or not a sample is present on the filter.

16. A method of nucleic acid extraction, characterized by, Comprising: a step of injecting a sample from a sample injection port and moving the sample along a flow path that communicates with a biological material collection region of a filter section to the biological material collection region to collect biological material containing nucleic acid on a filter of the filter section that is hydrophilic; a step of moving liquid that has passed through the filter along a flow path that communicates with a sample passage region of the filter section to a sample discharge port to discharge the liquid; a step of injecting a nucleic acid extraction solution from an opening that communicates with the biological material collection region and moving the nucleic acid extraction solution along a flow path that communicates with the biological material collection region to the biological material collection region to extract nucleic acid on the filter; and a step of recovering a solution containing the extracted nucleic acid on the filter from the biological material collection region through a flow path that communicates with the biological material collection region.

17. The nucleic acid extraction method according to claim 16, wherein The nucleic acid extraction solution is brought into contact with a portion of the filter and the nucleic acid extraction solution is moved in the flow path of the biological material collection region in such a manner that the entire filter is brought into contact with the nucleic acid extraction solution, whereby extraction of nucleic acid on the filter is performed.

18. A method of nucleic acid extraction, characterized by, Comprising: a step of injecting a sample from a sample injection port and moving the sample along a first flow path that communicates with a biological material collection region of a filter section to the biological material collection region to collect biological material containing nucleic acid on a filter of the filter section that is hydrophilic; a step of moving liquid that has passed through the filter along a second flow path that communicates with a sample passage region of the filter section to a sample discharge port to discharge the liquid; a step of injecting a nucleic acid extraction solution from the sample injection port or an air communication port that communicates with the biological material collection region and moving the nucleic acid extraction solution along a first flow path or a third flow path that communicates with the biological material collection region to the biological material collection region of the filter section to extract nucleic acid on the filter; a step of moving a solution containing the extracted nucleic acid on the filter to the third flow path that communicates with the biological material collection region; and a step of recovering the solution containing the extracted nucleic acid from the biological material collection region through the third flow path. a step of moving the solution containing the nucleic acid to a nucleic acid extraction liquid outlet by injecting air into the third flow path.

19. The nucleic acid extraction method according to claim 18, wherein, the nucleic acid extraction liquid is brought into contact with a portion of the filter and the nucleic acid extraction liquid is moved in the flow path of the biomaterial collection region in a manner that the entire filter is brought into contact with the nucleic acid extraction liquid, whereby extraction of the nucleic acid on the filter is performed.

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