Methods and apparatus for nucleic acid extraction and detection

Abstract

The present invention relates to a nucleic acid extraction member, which is manufactured from one or more of polyacrylate, polyamide, polyester, polyurethane, and polylactic acid, adsorbs nucleic acids in a solution with a pH of 2 to 6, and releases nucleic acids in a solution with a pH of 8 to 12, so that it can be used for nucleic acid extraction and detection, greatly shortening the time for nucleic acid extraction, washing, and purification, simplifying the operation procedures for nucleic acid extraction and purification, and reducing the consumption of equipment for nucleic acid extraction and purification.

Description

Technical Field

[0001] This application claims the priority of a prior application filed with the China National Intellectual Property Administration on July 22, 2022, with an application number of 202210874566.1 and a title of invention "Methods and Apparatus for Nucleic Acid Extraction and Detection". All contents of the prior application are incorporated herein by reference.

[0002] The present invention relates to the technical field of molecular biology, and more particularly to methods and apparatus for nucleic acid extraction and detection.

Background Art

[0003] In recent years, molecular biology diagnostic techniques based on nucleic acid detection have attracted attention. In techniques such as polymerase chain reaction (PCR), nucleic acids are the basis for biological detection, and improving the efficiency of nucleic acid extraction and shortening the time of nucleic acid extraction are the focus of attention of researchers.

[0004] Among current nucleic acid extraction methods, the classical phenol-chloroform extraction method needs to use organic solvents such as highly toxic phenol-chloroform for extraction, with complex operations and unstable extraction results, so it is hardly used in clinical genetic tests. The manual column passing method has a low detection leakage rate and high sensitivity, but it takes time, especially when the sample volume is large, the time cost increases significantly, so it is not suitable for screening a large number of samples. The one-step method (directly releasing nucleic acids in the sample using a sample releasing agent) has simple procedures but a high detection leakage rate and is not suitable for clinical use. Nucleic acids extracted by the magnetic bead method have high purity, can be used in combination with an automatic nucleic acid extraction device, have high extraction efficiency, and are suitable for the extraction of a large number of samples. However, the extraction procedure of the magnetic bead method nucleic acid extraction kit is complex, requires the use of an external magnetic field, takes at least 20 minutes to obtain nucleic acids in the laboratory, and it is difficult to meet the need for rapid nucleic acid detection. In addition, magnetic beads need to be stored at 4°C, and a large number of disposable pipette tips are generated during the extraction process. Therefore, there is an urgent need to develop a method and device for rapid, easy-to-use, and low-cost nucleic acid extraction and detection.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention provides a method and related products for nucleic acid extraction and detection, which solve the technical problems of the existing magnetic bead method, such as complex operation procedures, long required time, high requirements for temperature, the need to apply an external magnetic field, and consumption of consumables.

Means for Solving the Problems

[0006] The process of extracting nucleic acids by the existing magnetic bead method generally involves sampling → sending the sample to the laboratory for nucleic acid extraction and purification using a nucleic acid extraction kit → nucleic acid detection (specifically shown in Figure 1). Here, the procedures for nucleic acid extraction and purification performed in the laboratory are cumbersome, requiring lysis, adsorption, washing, and elution, taking at least 20 minutes for one sample, and even longer when multiple samples are operated simultaneously. The process of extracting nucleic acids of the present invention is sampling → immediately starting nucleic acid extraction after sampling → sending the sample to the laboratory for nucleic acid purification → nucleic acid detection (specifically shown in Figure 2). Such a "extract immediately after sampling" method makes full use of the time from sampling to sending the sample to the laboratory (usually at least 20 minutes), allowing nucleic acid purification to start after the sample arrives at the laboratory. The purification procedure takes only about 4 minutes, the whole process does not require other consumables such as low temperature, external magnetic field, pipette tips, etc., and at the same time, there is no need to add any additional reagents, and the nucleic acid yield is equivalent to that of the mainstream magnetic bead method currently on the market.

[0007] The present invention provides a nucleic acid extraction member made of one or more of polyacrylate, polyamide, polyester, polyurethane, and polylactic acid, which adsorbs nucleic acids in a first solution with a pH of 2 to 6, preferably 3 to 4, and releases nucleic acids in a second solution with a pH of 8 to 12, preferably 10 to 11. The nucleic acid is DNA and / or RNA and / or a fragment thereof.

[0008] As can be understood by those skilled in the art, various polyacrylates, polyamides, polyesters, polyurethanes, and polylactic acids can all be used in the present invention. In a preferred embodiment, the polyacrylate is methyl polymethacrylate, etc., and the polyester is polyethylene terephthalate, etc.

[0009] As can be understood by those skilled in the art, the nucleic acid extraction member of the present invention can have any shape, including regular or irregular shapes such as sheet-like, layered, spherical, ellipsoidal, cylindrical, frustum of a cone, conical, pyramidal, prismatic, frustum of a pyramid, cubic, or cuboid shapes.

[0010] The nucleic acid extraction member of the present invention can be used for the extraction and purification of nucleic acids, and the extracted and purified nucleic acids can be further used for nucleic acid detection. As can be understood by those skilled in the art, the nucleic acid extraction member of the present invention can be used for the detection of nucleic acids of microorganisms (such as viruses, bacteria, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes) in the human body, animal body, or environment (such as the surface of articles or foods, soil, air, etc.), and can also be used for the detection of disease-related genes (such as cancer-related genes, hereditary disease-related genes, etc.) in the human body or animal body.

[0011] Those skilled in the art can determine the volume and surface area of the nucleic acid extraction member according to the specific use of the nucleic acid extraction member, such as the amount of extracted nucleic acid and the amount of nucleic acid required for nucleic acid detection. For example, the volume of the nucleic acid extraction member is 0.1 to 2 cm 3 , preferably 0.3 to 1 cm 3 , more preferably 0.4 to 0.6 cm 3 , and for example, the surface area of the nucleic acid extraction member is 5 cm 2 or more, preferably 10 to 25 cm 2 , more preferably 13 to 16 cm 2 .

[0012] As can be understood by those skilled in the art, the nucleic acid extraction member of the present invention can be manufactured by methods known in this field. For example, fibers (such as monofilament fibers, etc.) can be knitted, needle-punched or wound into single-layer or multi-layer fabrics (such as mesh fabrics, non-woven fabrics, etc.), spheres, ellipsoids, cylinders, truncated cones, cones, pyramids, prisms, truncated pyramids, cubes, cuboids, etc. Alternatively, fibers (such as monofilament fibers, etc.) can first be knitted, needle-punched or wound into single-layer or multi-layer mesh fabrics, and then further manufactured into sheets, layers, spheres, ellipsoids, cylinders, truncated cones, cones, pyramids, prisms, truncated pyramids, cubes, cuboids, etc. by welding (such as heating and melting), or manufactured into porous sheets, layers, spheres, ellipsoids, cylinders, truncated cones, cones, pyramids, prisms, truncated pyramids, cubes, cuboids, etc. by foaming, etc.

[0013] As can be understood by those skilled in the art, the nucleic acid extraction member of the present invention may exist and be used alone, or may be fixed to a support member by winding and / or welding (such as heating and melting) and used. It may wrap the entire support member or a part of the support member. For example, it may be arranged or wrapped at one end of a rod-shaped support member. Also, short fibers can be fixed to one end of a rod-shaped support member by welding (such as heating and melting) to form a brush shape.

[0014] Preferably, the nucleic acid extraction member is arranged or wrapped at one end of a rod-shaped support member. For example, fibers (such as monofilament fibers, etc.) are fixed to one end of a rod-shaped support member by winding and / or welding (such as heating and melting), or first the fibers (such as monofilament fibers, etc.) are knitted, needle-punched or wound into a single-layer or multi-layer fabric, and then fixed to one end of a rod-shaped support member by winding and / or welding (such as heating and melting). Preferably, the length of the nucleic acid extraction member in the direction along the rod-shaped support member is 1 to 2 cm. Preferably, the diameter of the fiber is 0.05 to 0.5 mm, preferably 0.1 to 0.3 mm. Preferably, the rod-shaped support member has a point that is easy to break at one end close to the nucleic acid extraction member.

[0015] The present invention further provides a nucleic acid extraction method using the nucleic acid extraction member of the present invention.

[0016] The present invention further provides a nucleic acid detection method using the nucleic acid extraction member of the present invention.

[0017] As can be understood by those skilled in the art, the nucleic acid extraction method and detection method of the present invention do not necessarily aim at the diagnosis of diseases.

[0018] Preferably, the nucleic acid extraction method or nucleic acid detection method includes a step of placing a sample in a first solution with a pH of 2 to 6, preferably 3 to 4, and allowing the nucleic acid extraction member to adsorb nucleic acid, and a step of allowing the nucleic acid extraction member to release nucleic acid in a second solution with a pH of 8 to 12, preferably 10 to 11.

[0019] Preferably, the nucleic acid extraction method or nucleic acid detection method includes a step of placing a sample in a first solution with a pH of 2 to 6, preferably 3 to 4, and allowing the nucleic acid extraction member to adsorb nucleic acid, a step of washing the nucleic acid extraction member in a third solution with a pH of 3 to 6, preferably 5 to 6, and a step of allowing the nucleic acid extraction member to release nucleic acid in a second solution with a pH of 8 to 12, preferably 10 to 11.

[0020] In some embodiments of the present invention, the first solution may be referred to as an extraction solution, and the nucleic acid extraction member can adsorb nucleic acid therein. In a preferred embodiment, a biological membrane such as a cell membrane is dissolved in the extraction solution, and the nucleic acid to be extracted or detected is released into the extraction solution and then adsorbed by the nucleic acid extraction member. The third solution may be referred to as a washing solution, which plays a role in washing the nucleic acid extraction member and removing impurities such as saccharides, proteins, and lipids. The second solution may be referred to as a purification solution, which plays a role in releasing the nucleic acid adsorbed on the nucleic acid extraction member from the nucleic acid extraction member.

[0021] Preferably, the first solution is a buffer solution includingAs can be understood by those skilled in the art, the pH of the buffer can be adjusted to 2-6, preferably 3-4, using a pH adjuster. Preferably, the buffer is selected from MES buffer, Tris buffer, acetic acid-sodium acetate buffer, and citric acid-sodium citrate buffer. Preferably, the concentration of the buffer is 5-30 mM, more preferably 10-25 mM.

[0022] Preferably, the first solution is a nucleic acid lysis solution. As can be understood by those skilled in the art, the pH of the nucleic acid lysis solution can be adjusted to 2-6, preferably 3-4, using a pH adjuster. As can be understood by those skilled in the art, when the nucleic acid to be extracted or detected is in a biological membrane such as a cell membrane, the nucleic acid lysis solution can dissolve the biological membrane such as the cell membrane and release the nucleic acid to be extracted or detected into the solution. As can be understood by those skilled in the art, any nucleic acid lysis solution commonly used in this field can be used in the present invention.

[0023] Preferably, the nucleic acid lysis solution of the present invention contains 0.5-1.5 M arginine, 0.2-1.2 M inorganic salt, and a buffer, and the pH is 2-6, preferably 3-4.

[0024] Preferably, the concentration of arginine is 0.6-1.2 M.

[0025] Preferably, the concentration of the inorganic salt is 0.5-1 M.

[0026] Preferably, the inorganic salt is selected from sodium chloride, potassium chloride, sodium citrate, and any mixture thereof.

[0027] Preferably, the concentration of the buffer is 5-30 mM, more preferably 10-25 mM.

[0028] Preferably, the buffer is selected from MES buffer, Tris buffer, acetic acid-sodium acetate buffer, and citric acid-sodium citrate buffer.

[0029] Preferably, the nucleic acid lysis solution of the present invention further contains a pH adjuster.

[0030] Preferably, the nucleic acid lysate of the present invention further contains a metal chelating agent. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (for example, disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the concentration of the metal chelating agent is 1 to 20 mM, preferably 5 to 10 mM.

[0031] Preferably, the nucleic acid lysate of the present invention consists of the above components.

[0032] Since the nucleic acid lysate of the present invention does not contain a surfactant and a guanidine salt, it reduces the risk of sample contamination and avoids the risk associated with the formation of precipitation from the guanidine salt at low temperatures.

[0033] Preferably, the second solution is a buffer including As can be understood by those skilled in the art, a pH adjuster can be used to adjust the pH of the buffer to 8 to 12, preferably 10 to 11. Preferably, the buffer is selected from DPBS buffer, Tris buffer, glycine-sodium hydroxide buffer, and disodium hydrogen phosphate-sodium hydroxide buffer. Preferably, the concentration of Tris buffer, glycine-sodium hydroxide buffer, and disodium hydrogen phosphate-sodium hydroxide buffer is 1 to 30 mM, preferably 5 to 20 mM.

[0034] Preferably, the third solution is a buffer includingAs can be understood by those skilled in the art, the pH of the buffer can be adjusted to 3 - 6, preferably 5 - 6, using a pH adjuster. Preferably, the buffer is selected from Tris buffer, MES buffer, acetic acid - sodium acetate buffer, and citric acid - sodium citrate buffer. Preferably, the concentration of the buffer is 1 - 20 mM, more preferably 5 - 15 mM. Preferably, the buffer further contains a metal chelating agent. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (such as disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the concentration of the metal chelating agent is 0.5 - 10 mM, more preferably 1 - 5 mM.

[0035] As can be understood by those skilled in the art, any of the pH adjusters commonly used in the art, such as aqueous sodium hydroxide solution, aqueous ammonia solution, aqueous hydrochloric acid solution, etc., can be used in the present invention.

[0036] According to the present invention, all of the adsorption, washing, and release of nucleic acids can be carried out at a temperature above 0°C to 70°C, preferably 4 - 60°C, more preferably at room temperature.

[0037] Generally, the adsorption of nucleic acids can be completed in just 10 minutes and will not affect the detection results up to one week. To complete the adsorption of nucleic acids, the container containing the first solution can be left standing, shaken, or vortexed.

[0038] Generally, the washing of the nucleic acid extraction member can be completed in just 20 s, preferably 30 s. Therefore, the washing time of the nucleic acid extraction member is at least 20 s, preferably at least 30 s, more preferably 30 s - 2 minutes. More preferably, to complete the washing, the container containing the third solution can be left standing, shaken, or vortexed.

[0039] Generally, the release of nucleic acid can be completed in as little as 2 minutes, preferably 3 minutes. Therefore, the time for nucleic acid release is at least 2 minutes, preferably at least 3 minutes, more preferably 3 - 5 minutes. More preferably, to complete the release of nucleic acid, the container containing the second solution can be left standing, shaken, or vortexed.

[0040] The present invention further provides a kit or set for nucleic acid extraction or detection, which includes the nucleic acid extraction member of the present invention.

[0041] Preferably, the above-mentioned kit or set further includes a first solution with a pH of 2 - 6, preferably 3 - 4, placed in a first container. More preferably, it includes a second solution with a pH of 8 - 12, preferably 10 - 11, placed in a second container. Even more preferably, it further includes a third solution with a pH of 3 - 6, preferably 5 - 6, placed in a third container. In some embodiments of the present invention, the first container may be called a sampling tube, the first solution may be called an extraction solution, and the nucleic acid extraction member can adsorb nucleic acid therein. In a preferred embodiment, biological membranes such as cell membranes are dissolved in the extraction solution, and the nucleic acid to be extracted or detected is released into the extraction solution and then adsorbed by the nucleic acid extraction member. The third container may be called a washing tube, the third solution may be called a washing solution, and it serves to wash the nucleic acid extraction member and remove impurities. The second container may be called a purification tube, the second solution may be called a purification solution, and it serves to release the nucleic acid adsorbed on the nucleic acid extraction member from the nucleic acid extraction member.

[0042] Preferably, the first solution is a buffer including As can be understood by those skilled in the art, the pH of the buffer can be adjusted to 2 - 6, preferably 3 - 4, using a pH adjuster. Preferably, the buffer is selected from MES buffer, Tris buffer, acetic acid - sodium acetate buffer, citric acid - sodium citrate buffer. Preferably, the concentration of the buffer is 5 - 30 mM, more preferably 10 - 25 mM.

[0043] Preferably, the first solution is a nucleic acid lysate. As can be understood by those skilled in the art, the pH of the nucleic acid lysate can be adjusted to 2-6, preferably 3-4, using a pH adjuster.

[0044] Preferably, the nucleic acid lysate of the present invention contains 0.5-1.5 M arginine, 0.2-1.2 M inorganic salt, and a buffer solution, and the pH is 2-6, preferably 3-4.

[0045] Preferably, the concentration of arginine is 0.6-1.2 M.

[0046] Preferably, the concentration of the inorganic salt is 0.5-1 M.

[0047] Preferably, the inorganic salt is selected from sodium chloride, potassium chloride, sodium citrate, and any mixture thereof.

[0048] Preferably, the concentration of the buffer solution is 5-30 mM, more preferably 10-25 mM.

[0049] Preferably, the buffer solution is selected from MES buffer solution, Tris buffer solution, acetic acid-sodium acetate buffer solution, and citric acid-sodium citrate buffer solution.

[0050] Preferably, the nucleic acid lysate of the present invention further contains a pH adjuster.

[0051] Preferably, the nucleic acid lysate of the present invention further contains a metal chelating agent. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (for example, disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the concentration of the metal chelating agent is 1-20 mM, preferably 5-10 mM.

[0052] Preferably, the nucleic acid lysate of the present invention consists of the above components.

[0053] Preferably, the second solution is a buffer solution includingAs can be understood by those skilled in the art, the pH of the buffer can be adjusted to 8 to 12, preferably 10 to 11, using a pH adjuster. Preferably, the buffer is selected from DPBS buffer, Tris buffer, glycine-sodium hydroxide buffer, and disodium hydrogen phosphate-sodium hydroxide buffer. Preferably, the concentration of Tris buffer, glycine-sodium hydroxide buffer, and disodium hydrogen phosphate-sodium hydroxide buffer is 1 to 30 mM, preferably 5 to 20 mM.

[0054] Preferably, the third solution is a buffer including As can be understood by those skilled in the art, the pH of the buffer can be adjusted to 3 to 6, preferably 5 to 6, using a pH adjuster. Preferably, the buffer is selected from Tris buffer, MES buffer, acetic acid-sodium acetate buffer, and citric acid-sodium citrate buffer. Preferably, the concentration of the buffer is 1 to 20 mM, more preferably 5 to 15 mM. Preferably, the buffer further contains a metal chelating agent. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (such as disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the concentration of the metal chelating agent is 0.5 to 10 mM, more preferably 1 to 5 mM.

[0055] In the present invention, the nucleic acid extraction member exists alone or is fixed to a support member. As can be understood by those skilled in the art, the nucleic acid extraction member (including the nucleic acid extraction member fixed to the support member) can be placed in the first solution or exist independently, and can be placed in the first solution before, simultaneously with, or after the sample is placed in the first solution.

[0056] Preferably, the nucleic acid extraction member is disposed at or wrapped around one end of the rod-shaped support member. For example, a fiber (e.g., a monofilament fiber) is wound and / or fixed to one end of the rod-shaped support member by welding (e.g., heating and melting), or a fiber (e.g., a monofilament fiber) is first woven into a single-layer or multi-layer fabric, needle-punched, or wound, and then fixed to one end of the rod-shaped support member by winding and / or welding (e.g., heating and melting). Preferably, the length of the nucleic acid extraction member in the direction along the rod-shaped support member is 1 to 2 cm. Preferably, the diameter of the fiber is 0.05 to 0.5 mm, preferably 0.1 to 0.3 mm.

[0057] Preferably, the other end of the rod-shaped support member is fixed inside the sealing members corresponding to the first container and the third container. In some embodiments of the present invention, the first container and the third container are tubular, and the sealing member is a tube cap. As can be understood by those skilled in the art, the rod-shaped support member can be fixed inside the sealing members corresponding to the first container and the third container by methods known in the art such as welding or screwing.

[0058] Preferably, the rod-shaped support member has a point that is easily breakable at one end close to the nucleic acid extraction member.

[0059] Preferably, the kit or set further includes a fourth container for packaging the nucleic acid extraction member. Preferably, the fourth container corresponds to the sealing member to which the rod-shaped support member is fixed. Preferably, the fourth container is tubular. Preferably, the fourth container contains no other contents and is used only for packaging the nucleic acid extraction member.

[0060] Preferably, the nucleic acid extraction member is placed in the first solution.

[0061] Preferably, the volume of the first solution is 1 to 10 mL, more preferably 1.5 to 3 mL, Preferably, the volume of the second solution is 100 to 500 μL, more preferably 150 to 350 μL, Preferably, the volume of the third solution is 1 to 10 mL, more preferably 2 to 4 mL.

[0062] As can be understood by those skilled in the art, the above kit or set further includes a sampling member. In some embodiments of the present invention, the sampling member is a throat swab. Of course, as can be understood by those skilled in the art, the rod-shaped support member to which the nucleic acid extraction member is fixed can be used simultaneously as a sampling member, for example, as a throat swab.

[0063] The present invention further provides the use of polyacrylate, polyamide, polyester, polyurethane or polylactic acid in nucleic acid extraction.

[0064] The present invention further provides the use of polyacrylate, polyamide, polyester, polyurethane or polylactic acid in the manufacture of a kit or set for nucleic acid extraction or nucleic acid detection. Preferably, the use is to manufacture the above nucleic acid extraction member.

Advantages of the Invention

[0065] The sample collection and nucleic acid extraction processes of the present invention can be carried out immediately sequentially or simultaneously, making full use of the time taken in the sample transportation process, and it is possible to complete the purification of nucleic acid within 4 minutes after the sample arrives at the laboratory. And the nucleic acid yield of the present invention is equivalent to that of the currently commercially available magnetic bead method.

Brief Description of the Drawings

[0066]

Figure 1

Figure 2

Embodiments for Carrying out the Invention

[0067] Hereinafter, the present invention will be further described with reference to specific examples. It should be understood that these examples are only for explaining the present invention and do not limit the scope of the present invention. Also, after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and it should be understood that these equivalent forms fall within the scope limited by the present invention.

[0068] The experimental methods used in the following examples are all ordinary methods unless otherwise specified. The reagents, materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0069] In the description of the present invention, it should be noted that terms such as "first", "second", "third", "fourth", "fifth", etc. are used only for descriptive distinction and do not indicate or imply the relative importance of the object.

[0070] An option indicates the presence or absence of the said feature, and also indicates that the said feature must be present, but specific selection can be freely made.

[0071] Example 1 Polyethylene terephthalate fiber (PET, Nantong New Dike Monofilament Technology Co., Ltd., diameter 0.15 mm, D0150) and polylactic acid fiber (PLA, Nantong New Dike Monofilament Technology Co., Ltd., diameter 0.25 mm, 0501) were respectively used as warp and weft to weave a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of this end. The other end of the rod was fixed inside the pipe cap corresponding to the sampling pipe and the washing pipe by welding or screwing. In this example, the volume of the nucleic acid extraction member made of polyethylene terephthalate fiber and polylactic acid fiber was about 0.4 cm 3 and the surface area was about 16 cm 2 at that time.

[0072] Preparation of Dissolution Solution, Washing Solution and Purification Solution:

[0073] A 10 mM MES buffer containing 0.6 M arginine, 0.5 M NaCl and 5 mM EDTA was prepared, and the pH value was adjusted to 3 with a 1 M aqueous sodium hydroxide solution and / or a 1 M aqueous hydrochloric acid solution to obtain a dissolution solution, which was dispensed into sampling tubes at 2 mL each.

[0074] A 5 mM Tris buffer containing 3 mM disodium EDTA was prepared, and the pH was adjusted to 3 with a 1 M aqueous sodium hydroxide solution and / or a 1 M aqueous hydrochloric acid solution to obtain a washing solution, which was dispensed into washing tubes at 2 mL each.

[0075] DPBS buffer was prepared, and the pH was adjusted to 9 with a 1 M aqueous sodium hydroxide solution and / or a 1 M aqueous hydrochloric acid solution to obtain a purification solution, which was dispensed into purification tubes at 300 μL each.

[0076] Method for Nucleic Acid Extraction and Detection:

[0077] 1. The H1N1 virus solution was serially diluted until the concentration reached 5×10 3 copies / mL.

[0078] 2. 222 μL of the virus solution was taken and added to a conventional inactivated sampling tube (Youkang Biotech (Beijing) Co., Ltd., MT0501-7-3) containing 2 mL of sampling solution to make the final concentration of H1N1 virus 5×10 2 copies / mL. Similarly, 222 μL of the virus solution was taken and added to the sampling tube manufactured in this example. After making the final concentration of H1N1 virus 5×10 2 copies / mL, the sampling tube was covered with the tube cap with a nucleic acid extraction member manufactured in this example, and the nucleic acid extraction member was completely immersed in the mixed solution of the virus solution and the dissolution solution in the sampling tube.

[0079] 3. After leaving the conventional inactivated sampling tube standing for 0.5 hours, nucleic acids were extracted according to the instructions using a magnetic bead-based nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100). Then, fluorescence quantitative PCR detection was performed, with three repetitions of Ct-1, Ct-2, and Ct-3 respectively, and the average value was taken. The results are shown in Table 1 below.

[0080] 4. After leaving the sampling tube of the present invention standing for 0.5 hours, the tube cap with a nucleic acid extraction member was covered on the washing tube, the nucleic acid extraction member was completely immersed in the washing solution in the washing tube, and after tightening, it was vertically shaken for about 30 s.

[0081] 5. Remove the tube cap with the nucleic acid extraction member, fold one end with the nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, then vortex for about 3 min, and then perform fluorescence quantitative PCR detection, with three repetitions of Ct-1, Ct-2, and Ct-3 respectively, and the average value was taken. The results are shown in Table 1 below.

[0082]

Table 1

[0083] As can be seen from this, the nucleic acids extracted by the method of the present invention have no significant difference in PCR detection compared with the magnetic bead method, but the method of the present invention is easy to operate, takes less time, and only takes about 4 min for nucleic acid washing and purification, while the magnetic bead method takes about 20 min for extraction, washing, and purification.

[0084] Example 2 One end of 200 polyurethane fibers (Nantong Xindike Monofilament Technology Co., Ltd., with a length of about 1 cm, a diameter of 0.2 mm, J0200) was welded within about 1.2 cm from the tip of one end of an ABS rod by heating and melting. The rod has a breakable point about 3 cm from the top of that end. The other end of the rod was fixed inside the tube cap corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of polyurethane fiber is about 0.5 cm 3 and the surface area is about 14 cm 2 .

[0085] Preparation of lysis solution, washing solution and purification solution:

[0086] Prepare a 15 mM MES buffer containing 0.8 M arginine, 1 M NaCl and 8 mM disodium EDTA, adjust the pH to 4 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a lysis solution, and dispense 2 mL aliquots into sampling tubes.

[0087] Prepare a 10 mM Tris buffer containing 1 mM disodium EDTA, adjust the pH to 4 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a washing solution, and dispense 2 mL aliquots into washing tubes.

[0088] Prepare DPBS buffer, adjust the pH to 11 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a purification solution, and dispense 300 μL aliquots into purification tubes.

[0089] Methods for nucleic acid extraction and detection:

[0090] 1. Dilute the H1N1 virus solution serially until the concentration reaches 5×10 3 copies / mL.

[0091] 2. Take 222 μL of the virus solution and add it to a conventional inactivated sampling tube (Youkang Biotechnology (Beijing) Co., Ltd., MT0501-7-3) containing 2 mL of sampling solution, so that the final concentration of H1N1 virus is 5×102 It was made into copies / mL. Similarly, 222 μL of the virus solution was taken and added to the sampling tube manufactured in this example, and the final concentration of the H1N1 virus was made 5×10 2 copies / mL. Then, the sampling tube was covered with the tube lid with a nucleic acid extraction member manufactured in this example, and the nucleic acid extraction member was completely immersed in the mixed solution of the virus solution and the lysis solution in the sampling tube.

[0092] 3. After allowing the conventional inactivated sampling tube to stand for 0.5 hour, nucleic acid was extracted using a magnetic bead method nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102 - 100) according to its instructions, and then fluorescence quantitative PCR detection was performed. It was repeated 3 times for Ct - 1, Ct - 2, and Ct - 3 respectively, and the average value was taken. The results are shown in Table 2 below.

[0093] 4. After allowing the sampling tube of the present invention to stand for 0.5 hour, the tube lid with a nucleic acid extraction member was covered with a washing tube, the nucleic acid extraction member was completely immersed in the washing solution in the washing tube, and after tightening, it was vertically shaken for about 30 s.

[0094] 5. Remove the tube lid with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube lid, then vortex - vibrate for about 3 min, and then perform fluorescence quantitative PCR detection. It was repeated 3 times for Ct - 1, Ct - 2, and Ct - 3 respectively, and the average value was taken. The results are shown in Table 2 below.

[0095]

Table 2

[0096] Example 3 Poly lactic acid fiber (PLA, Nantong Xindike Monofilament Technology Co., Ltd., diameter 0.25 mm, 0501) was knitted into a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of the end. The other end of the rod was fixed inside the tube cap corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of poly lactic acid fiber was about 0.5 cm 3 and the surface area was about 13 cm 2 .

[0097] Preparation of lysis solution, washing solution and purification solution:

[0098] A 25 mM acetic acid-sodium acetate buffer containing 0.6 M arginine, 1.2 M NaCl and 5 mM disodium EDTA was prepared, and the pH was adjusted to 6 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a lysis solution, which was dispensed into sampling tubes at 2 mL each.

[0099] A 15 mM Tris buffer containing 5 mM disodium EDTA was prepared, and the pH was adjusted to 6 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a washing solution, which was dispensed into washing tubes at 2 mL each.

[0100] A 5 mM Tris buffer was prepared, and the pH was adjusted to 8 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a purification solution, which was dispensed into purification tubes at 300 μL each.

[0101] Method for nucleic acid extraction and detection:

[0102] 1. The H1N1 virus solution was serially diluted until the concentration reached 5×10 3 copies / mL.

[0103] 2. Take 222 μL of the virus solution and add it to a conventional inactivated sampling tube (Youkang Biotechnology (Beijing) Co., Ltd., MT0501-7-3) containing 2 mL of sampling solution, so that the final concentration of H1N1 virus is 5×10 2 copies / mL. Similarly, take 222 μL of the virus solution, add it to the sampling tube manufactured in this example, and after making the final concentration of H1N1 virus 5×10 2 copies / mL, cover the sampling tube with the tube cap with a nucleic acid extraction member manufactured in this example, and completely immerse the nucleic acid extraction member in the mixed solution of the virus solution and the lysis solution in the sampling tube.

[0104] 3. After leaving the conventional inactivated sampling tube standing for 0.5 h, use a magnetic bead method nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100) to extract nucleic acid according to its instruction manual, and then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2, and Ct-3 respectively, take the average value, and the results are shown in Table 3 below.

[0105] 4. After leaving the sampling tube of the present invention standing for 0.5 h, cover the tube cap with a nucleic acid extraction member with a cleaning tube, completely immerse the nucleic acid extraction member in the cleaning solution in the cleaning tube, tighten it, and then vibrate vertically for about 30 s.

[0106] 5. Remove the tube cap with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, and then vortex for about 3 min. Then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2, and Ct-3 respectively, take the average value, and the results are shown in Table 3 below.

[0107]

Table 3

[0108] Example 4 Poly(methyl methacrylate) (PMMA) fibers (manufactured by Shenzhen Chuangli Optoelectronic Fiber Optics Materials Co., Ltd., with a diameter of 0.2 mm, CL-250) were knitted into a mesh fabric with a width of approximately 2 cm, cut into a rectangle with a length of approximately 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is approximately 2 cm, and the rod has a point where it is prone to breakage approximately 3 cm from the tip of this end. The other end of the rod was fixed inside the tube lid corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of poly(methyl methacrylate) fibers is approximately 0.6 cm 3 and the surface area is approximately 16 cm 2 .

[0109] Preparation of lysis solution, washing solution and purification solution:

[0110] Prepare a 10 mM acetic acid-sodium acetate buffer containing 1 M arginine, 0.5 M KCl and 10 mM EDTA, adjust the pH value to 6 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain the lysis solution, and dispense 2 mL each into the sampling tubes.

[0111] Prepare a 5 mM MES buffer containing 5 mM disodium EDTA, adjust the pH to 4 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain the washing solution, and dispense 2 mL each into the washing tubes.

[0112] Prepare a 10 mM Tris buffer, adjust the pH to 9 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain the purification solution, and dispense 300 μL each into the purification tubes.

[0113] Methods for nucleic acid extraction and detection:

[0114] 1. The H1N1 virus solution was serially diluted until the concentration reached 5×10 3 copies / mL.

[0115] 2. Take 222 μL of the virus solution and add it to a conventional inactivated sampling tube (Youkang Biotechnology (Beijing) Co., Ltd., MT0501-7-3) containing 2 mL of sampling solution, so that the final concentration of H1N1 virus is 5×10 2 copies / mL. Similarly, take 222 μL of the virus solution, add it to the sampling tube manufactured in this example, and after making the final concentration of H1N1 virus 5×10 2 copies / mL, cover the sampling tube with the tube cap with a nucleic acid extraction member manufactured in this example, and completely immerse the nucleic acid extraction member in the mixed solution of the virus solution and the lysis solution in the sampling tube.

[0116] 3. After leaving the conventional inactivated sampling tube standing for 0.5 hour, use a magnetic bead method nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100) to extract nucleic acids according to its instructions, and then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2, and Ct-3 respectively, take the average value, and the results are shown in Table 4 below.

[0117] 4. After leaving the sampling tube of the present invention standing for 0.5 hour, cover the tube cap with a nucleic acid extraction member with a cleaning tube, completely immerse the nucleic acid extraction member in the cleaning solution in the cleaning tube, tighten it, and then shake it vertically for about 30 s.

[0118] 5. Remove the tube cap with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, then vortex for about 3 min, and then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2, and Ct-3 respectively, take the average value, and the results are shown in Table 4 below.

[0119]

Table 4

[0120] Example 5 PMMA fibers (manufactured by Shenzhen Chuangli Optoelectronic Fiber Optics Materials Co., Ltd., with a diameter of 0.2 mm, CL-250) were knitted into a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of this end. The other end of the rod was fixed inside the tube cap corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of PMMA fibers is about 0.4 cm 3 and the surface area is about 14 cm 2 .

[0121] Preparation of lysis solution, washing solution and purification solution:

[0122] Prepare a 15 mM MES buffer containing 0.8 M arginine, 1.0 M KCl and 8 mM EDTA, adjust the pH value to 3 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a lysis solution, and dispense 2 mL each into the sampling tubes.

[0123] Prepare a 10 mM MES buffer containing 1 mM disodium EDTA, adjust the pH to 6 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a washing solution, and dispense 2 mL each into the washing tubes.

[0124] Prepare a 15 mM Tris buffer, adjust the pH to 10 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a purification solution, and dispense 300 μL each into the purification tubes.

[0125] Method for nucleic acid extraction and detection:

[0126] 1. Culture MDCK cells until the confluence reaches 90%, digest them with EDTA trypsin, collect them in a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant to obtain cell precipitate, add 1 mL of PBS to resuspend the cells, and count the cell suspension. The density was 1×10 7 / mL.

[0127] 2. Take 222 μL of the cell suspension, add it to the sampling tube manufactured in this example, and then cover the sampling tube with the tube cap with a nucleic acid extraction member manufactured in this example, completely immersing the nucleic acid extraction member in the mixed solution of the cell suspension and the lysis solution in the sampling tube.

[0128] 3. After shaking at 150 rpm for 0.5 h on a shaker at 37 °C, cover the tube cap with the nucleic acid extraction member with a washing tube, completely immerse the nucleic acid extraction member in the washing solution in the washing tube, tighten it, and then shake vertically for about 30 s.

[0129] 4. Remove the tube cap with the nucleic acid extraction member, fold one end with the nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, then vortex for about 3 min, and then detect the nucleic acid content using an ultra-micro ultraviolet spectrophotometer. The results are shown in Table 5 below.

[0130] Take 222 μL of the cell suspension, add it to a conventional inactivated sampling tube (Youkang Biotechnology (Beijing) Co., Ltd., MT0501-7-3) containing 2 mL of sampling solution, let it stand for 0.5 h, and then use a magnetic bead method nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100) to extract nucleic acid according to the instructions. Then, detect the nucleic acid content using an ultra-micro ultraviolet spectrophotometer. The results are shown in Table 5 below.

[0131]

Table 5

[0132] Example 6 Polyamide (PA66) fibers (Nantong Xindike Monofilament Technology Co., Ltd., diameter 0.25 mm, J250D) were knitted into a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of this end. The other end of the rod was fixed inside the tube cap that can correspond to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of polyamide is about 0.4 cm 3 and the surface area is about 16 cm 2 .

[0133] Preparation of lysis solution, washing solution and purification solution:

[0134] Prepare a 25 mM acetic acid-sodium acetate buffer containing 0.5 M arginine, 1.2 M KCl and 6 mM EDTA, adjust the pH to 4 with 1 M sodium hydroxide aqueous solution and / or 1 M hydrochloric acid aqueous solution to obtain a lysis solution, and dispense 2 mL each into the sampling tubes.

[0135] Prepare a 15 mM MES buffer containing 5 mM disodium EDTA, adjust the pH to 5 with 1 M sodium hydroxide aqueous solution and / or 1 M hydrochloric acid aqueous solution to obtain a washing solution, and dispense 2 mL each into the washing tubes.

[0136] Prepare a 5 mM glycine-sodium hydroxide buffer, adjust the pH to 10 with 1 M sodium hydroxide aqueous solution and / or 1 M hydrochloric acid aqueous solution to obtain a purification solution, and dispense 300 μL each into the purification tubes.

[0137] Methods for nucleic acid extraction and detection:

[0138] 1. Dilute the H1N1 virus solution by gradient until the concentration reaches 5×10 3 copies / mL.

[0139] 2. Take 222 μL of the virus solution and add it to a conventional inactivated sampling tube (Youkang Biotech (Beijing) Co., Ltd., MT0501-7-3) containing 2 mL of sampling solution, so that the final concentration of H1N1 virus is 5×10 2 copies / mL. Similarly, take 222 μL of the virus solution, add it to the sampling tube manufactured in this example, and after setting the final concentration of H1N1 virus to 5×10 2 copies / mL, cover the sampling tube with the tube cap with a nucleic acid extraction member manufactured in this example, and completely immerse the nucleic acid extraction member in the mixed solution of the virus solution and the lysis solution in the sampling tube.

[0140] 3. After leaving the conventional inactivated sampling tube standing for 0.5 hour, use a magnetic bead method nucleic acid extraction kit (Youkang Biotech (Beijing) Co., Ltd., MK0102-100) to extract nucleic acids according to its instructions, and then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2, and Ct-3 respectively, take the average value, and the results are shown in Table 6 below.

[0141] 4. After leaving the sampling tube of the present invention standing for 0.5 hour, cover the tube cap with a nucleic acid extraction member with a washing tube, completely immerse the nucleic acid extraction member in the washing solution in the washing tube, tighten it, and then shake it vertically for about 30 s.

[0142] 5. Remove the tube cap with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, then vortex for about 3 min, and then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2, and Ct-3 respectively, take the average value, and the results are shown in Table 6 below.

[0143]

Table 6

[0144] Example 7 Polyethylene terephthalate fiber (PET, Nantong New Dike Monofilament Technology Co., Ltd., diameter 0.15 mm, D0150) and polylactic acid fiber (PLA, Nantong New Dike Monofilament Technology Co., Ltd., diameter 0.25 mm, 0501) were used as warp and weft respectively to weave a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of this end. The other end of the rod was fixed inside the tube cap corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of polyethylene terephthalate fiber and polylactic acid fiber is about 0.4 cm 3 and the surface area is about 16 cm 2 was.

[0145] Preparation of lysis solution, washing solution and purification solution:

[0146] A 10 mM MES buffer containing 0.5 M arginine, 0.5 M sodium citrate and 6 mM EDTA was prepared, and the pH value was adjusted to 5 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a lysis solution, which was dispensed into sampling tubes at 2 mL each.

[0147] A 10 mM acetic acid - sodium acetate buffer containing 2 mM disodium EDTA was prepared, and the pH was adjusted to 3 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a washing solution, which was dispensed into washing tubes at 2 mL each.

[0148] A 15 mM glycine - sodium hydroxide buffer was prepared, and the pH was adjusted to 11 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a purification solution, which was dispensed into purification tubes at 300 μL each.

[0149] Methods for nucleic acid extraction and detection:

[0150] 1. The PEDV virus solution was serially diluted until the concentration reached 5×10 3 copies / mL.

[0151] 2. Take 222 μL of the virus solution and add it to the sampling tube manufactured in this example to make the final concentration of the PEDV virus 5×10 2 copies / mL. Then, cover the sampling tube with the tube lid with a nucleic acid extraction member manufactured in this example, completely immerse the nucleic acid extraction member in the mixed solution of the virus solution and the lysis solution in the sampling tube, and repeat by setting up a total of 7 tubes.

[0152] 3. After leaving one of the tubes standing for 10 minutes, cover the tube lid with a nucleic acid extraction member with a cleaning tube, completely immerse the nucleic acid extraction member in the cleaning solution in the cleaning tube, tighten it, and then shake it vertically for about 30 seconds.

[0153] 4. Remove the tube lid with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube lid, and then vortex for about 3 minutes to obtain the purified nucleic acid. Store the purified nucleic acid frozen at -80°C and record it as the nucleic acid obtained by extraction on the first day.

[0154] 5. Leave the remaining 6 tubes standing until the 2nd to 7th days respectively, repeat the above steps "3, 4", and obtain the nucleic acids on the 2nd to 7th days respectively.

[0155] 6. Perform fluorescence quantitative PCR on the nucleic acids on the 1st to 7th days, repeat twice for Ct-1 and Ct-2 respectively, take the average value, compare whether there are significant differences at different times, and show the detection results in Table 7 below.

[0156]

Table 7

[0157] As can be seen from this, the method of the present invention has a short extraction time of 10 minutes and a long extraction time of 7 days. When the nucleic acid obtained by extraction is detected by PCR, there is no significant difference, indicating that the method of the present invention is very stable.

[0158] Example 8 The polylactic acid fiber (PLA, Nantong Xindi Ke Monofilament Technology Co., Ltd., diameter 0.25 mm, 0501) was knitted into a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of a rod made of ABS by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of the end. The other end of the rod was fixed inside a tube cap that can correspond to a sampling tube and a washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of polylactic acid fiber is about 0.4 cm 3 and the surface area is about 16 cm 2 .

[0159] Preparation of lysis solution, washing solution and purification solution:

[0160] A 15 mM citric acid-sodium citrate buffer containing 0.7 M arginine, 1 M sodium citrate and 8 mM disodium EDTA was prepared, and the pH was adjusted to 2 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a lysis solution, which was dispensed into sampling tubes at 2 mL each.

[0161] A 15 mM acetic acid-sodium acetate buffer containing 5 mM disodium EDTA was prepared, and the pH was adjusted to 5 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a washing solution, which was dispensed into washing tubes at 2 mL each.

[0162] A 20 mM glycine-sodium hydroxide buffer was prepared, and the pH was adjusted to 12 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain a purification solution, which was dispensed into purification tubes at 300 μL each.

[0163] Methods for nucleic acid extraction and detection:

[0164] 1. The PEDV virus solution was serially diluted until the concentration reached 5×10 4 copies / mL.

[0165] 2. Take 222 μL of the virus solution and add it to the sampling tube manufactured in this example. After setting the final concentration of the PEDV virus to 5×10 2 copies / mL, cover the sampling tube with the tube cap with a nucleic acid extraction member manufactured in this example, completely immerse the nucleic acid extraction member in the mixed solution of the virus solution and the lysis solution in the sampling tube, and repeat by setting up 4 tubes.

[0166] 3. Place the 4 tubes in environments of 4°C, 25°C, 37°C, and 60°C respectively, and shake at 150 rpm for 0.5 hours.

[0167] 4. After 0.5 hours, cover the tube cap with a nucleic acid extraction member of each group with a washing tube, completely immerse the nucleic acid extraction member in the washing solution in the washing tube, tighten it, and then shake vertically for about 30 s.

[0168] 5. Remove the tube cap with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the breaking point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, then vortex for about 3 min, and then perform fluorescence quantitative PCR detection, repeat 2 times for Ct-1 and Ct-2 respectively, take the average value, and the results are shown in Table 8 below.

[0169]

Table 8

[0170] As can be seen from this, the method of the present invention shows that the extraction temperature is short at 4°C and long at 60°C, and there is no significant difference in detecting the nucleic acid obtained by extraction by PCR, indicating that the method of the present invention is very stable.

[0171] Example 9 Methyl polymethacrylate (PMMA) fiber (Chuangli Optoelectronic Fiber Optics Materials Co., Ltd., Shenzhen, diameter 0.2 mm, CL-250) and polylactic acid fiber (PLA, Nantong Xindike Monofilament Technology Co., Ltd., diameter 0.25 mm, 0501) were used as warp and weft respectively to weave a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of this end. The other end of the rod was fixed inside the tube cap corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of methyl polymethacrylate fiber and polylactic acid fiber is about 0.5 cm 3 and the surface area is about 13 cm 2 was.

[0172] Preparation of lysis solution, washing solution and purification solution:

[0173] Prepare a 25 mM citric acid-sodium citrate buffer containing 0.7 M arginine, 1.2 M sodium citrate and 10 mM EDTA, adjust the pH to 6 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain the lysis solution, and dispense 2 mL each into the sampling tubes.

[0174] Prepare a 15 mM citric acid-sodium citrate buffer containing 5 mM disodium EDTA, adjust the pH to 6 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain the washing solution, and dispense 2 mL each into the washing tubes.

[0175] Prepare a 5 mM disodium hydrogen phosphate-sodium hydroxide buffer, adjust the pH to 10 with 1 M aqueous sodium hydroxide solution and / or 1 M aqueous hydrochloric acid solution to obtain the purification solution, and dispense 300 μL each into the purification tubes.

[0176] Methods for nucleic acid extraction and detection:

[0177] 1. The H1N1 virus solution was adjusted to a concentration of 5×10 3It was serially diluted until it reached the copy number per mL.

[0178] 2. Take two portions of 222 μL of the virus solution and add them to conventional inactivated sampling tubes (Youkang Biotechnology (Beijing) Co., Ltd., MT0501-7-3 and MT0501-2, both containing guanidine salts in the lysate) each containing 2 mL of the sampling solution, so that the final concentration of H1N1 virus was 5×10 2 copies / mL. Similarly, take 222 μL of the virus solution, add it to the sampling tube manufactured in this example, and after setting the final concentration of H1N1 virus to 5×10 2 copies / mL, cover the above three sampling tubes with the tube caps with nucleic acid extraction members manufactured in this example, and completely immerse the nucleic acid extraction members in the mixed solution of the virus solution and the lysate in the sampling tubes.

[0179] 3. After leaving the above three sampling tubes standing for 0.5 hour, cover the tube caps with nucleic acid extraction members with a washing tube, completely immerse the nucleic acid extraction members in the washing solution in the washing tube, tighten it, and then vertically shake it for about 30 s.

[0180] 4. Remove the tube caps with nucleic acid extraction members, fold one end with the nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube cap, then vortex for about 3 min, and then perform fluorescence quantitative PCR detection. Repeat three times for Ct-1, Ct-2 and Ct-3 respectively, take the average value, and the results are shown in Table 9 below.

[0181]

Table 9

[0182] As can be seen from this, the method of the present invention can effectively detect nucleic acids even when using various lysates, and the effect of the lysate containing no guanidine salt of the present invention is superior to that of the lysate containing guanidine salt.

[0183] Example 10 Polyamide (PA66) fiber (Nantong New Dike Monofilament Technology Co., Ltd., diameter 0.25 mm, J250D) and polylactic acid fiber (PLA, Nantong New Dike Monofilament Technology Co., Ltd., diameter 0.25 mm, 0501) were used as warp and weft respectively to weave a mesh fabric with a width of about 2 cm, cut into a rectangle with a length of about 5 cm, wound, and welded to one end of an ABS rod by heating and melting. The length is about 2 cm, and the rod has a breakable point about 3 cm from the tip of this end. The other end of the rod was fixed inside the tube cap corresponding to the sampling tube and the washing tube by welding or screwing. In this example, the volume of the nucleic acid extraction member made of polyamide fiber and polylactic acid fiber is about 0.6 cm 3 and the surface area is about 16 cm 2 was.

[0184] Preparation of lysis solution, washing solution and purification solution:

[0185] Prepare a 25 mM acetic acid-sodium acetate buffer containing 0.8 M arginine, 0.5 M NaCl and 5 mM EDTA, adjust the pH to 4 with 1 M aqueous sodium hydroxide solution and / or 1 M hydrochloric acid aqueous solution to obtain a lysis solution, and dispense 2 mL each into sampling tubes.

[0186] Prepare a 5 mM citric acid-sodium citrate buffer containing 2 mM disodium EDTA, adjust the pH to 4 with 1 M aqueous sodium hydroxide solution and / or 1 M hydrochloric acid aqueous solution to obtain a washing solution, and dispense 2 mL each into washing tubes.

[0187] Prepare a 5 mM disodium hydrogen phosphate-sodium hydroxide buffer, adjust the pH to 11 with 1 M aqueous sodium hydroxide solution and / or 1 M hydrochloric acid aqueous solution to obtain a purification solution, and dispense 2 mL each into purification tubes.

[0188] Methods for nucleic acid extraction and detection:

[0189] 1. Dilute the PEDV virus solution serially until the concentration reaches 5×10 3 copies / mL.

[0190] 2. Take 2 parts of 222 μL of the virus solution and add them to conventional inactivation sampling tubes (Youkang Biotech (Beijing) Co., Ltd., MT0501-7-3 and MT0501-2, both of which contain guanidine salts in the lysate) containing 2 mL of sampling solution each, so that the final concentration of PEDV virus is 5×10 2 copies / mL. Similarly, take 222 μL of the virus solution, add it to the sampling tube manufactured in this example, and after setting the final concentration of PEDV virus to 5×10 2 copies / mL, cover the above three sampling tubes with the tube lid with a nucleic acid extraction member manufactured in this example, and completely immerse the nucleic acid extraction member in the mixed solution of the virus solution and the lysate in the sampling tube.

[0191] 3. After leaving the above three sampling tubes standing for 0.5 hour, cover the tube lid with a nucleic acid extraction member with a washing tube, completely immerse the nucleic acid extraction member in the washing solution in the washing tube, tighten it, and then shake it vertically for about 30 s.

[0192] 4. Remove the tube lid with a nucleic acid extraction member, fold one end with a nucleic acid extraction member at the folding point of the rod so as to fold it into the purification tube, completely immerse the nucleic acid extraction member in the purification solution of the purification tube, tighten the tube lid, then vortex for about 3 min, and then perform fluorescence quantitative PCR detection, repeat it 3 times for Ct-1, Ct-2 and Ct-3 respectively, take the average value, and the results are shown in Table 10 below.

[0193]

Table 10

[0194] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid extraction member, which is made of one or more of polyacrylate, polyamide, polyester, polyurethane and polylactic acid, adsorbs nucleic acid in a first solution having a pH of 2 to 6, preferably 3 to 4, and releases nucleic acid in a second solution having a pH of 8 to 12, preferably 10 to 11. Preferably, the nucleic acid is DNA and / or RNA and / or a fragment thereof. Preferably, the polyacrylate is methyl polymethacrylate. Preferably, the polyester is polyethylene terephthalate. Preferably, the volume of the nucleic acid extraction member is 0.1 to 2 cm ３ , more preferably 0.3 to 1 cm ３ , even more preferably 0.4 to 0.6 cm ３ and Preferably, the surface area of the nucleic acid extraction member is 5 cm ２ or more, more preferably 10 to 25 cm ２ , even more preferably 13 to 16 cm ２ and Preferably, the nucleic acid extraction member is disposed at or wrapped around one end of a rod-shaped support member. Preferably, the length of the nucleic acid extraction member in the direction along the rod-shaped support member is 1 to 2 cm. Preferably, the rod-shaped support member has a point that is easily breakable at one end close to the nucleic acid extraction member. A nucleic acid extraction member characterized by this.

2. A nucleic acid extraction method, characterized by using the nucleic acid extraction member according to Claim 1.

3. A nucleic acid detection method, characterized by using the nucleic acid extraction member according to Claim 1.

4. Including the procedure of putting the sample into a first solution having a pH of 2 to 6, preferably 3 to 4, and the nucleic acid extraction member adsorbing the nucleic acid, and the procedure of the nucleic acid extraction member releasing the nucleic acid in a second solution having a pH of 8 to 12, preferably 10 to 11. Preferably, including the procedure of putting the sample into a first solution having a pH of 2 to 6, preferably 3 to 4, and the nucleic acid extraction member adsorbing the nucleic acid, the procedure of washing the nucleic acid extraction member in a third solution having a pH of 3 to 6, preferably 5 to 6, and the procedure of the nucleic acid extraction member releasing the nucleic acid in a second solution having a pH of 8 to 12, preferably 10 to 11. Preferably, the first solution is a buffer solution. More preferably, the buffer solution is selected from MES buffer solution, Tris buffer solution, acetic acid - sodium acetate buffer solution, citric acid - sodium citrate buffer solution. Preferably, the concentration of the buffer solution is 5 to 30 mM, more preferably 10 to 25 mM. Preferably, the first solution is a nucleic acid lysate. Preferably, the nucleic acid lysate contains 0.5 to 1.5 M arginine, 0.2 to 1.2 M inorganic salt, and a buffer solution, and the pH is 2 to 6, preferably 3 to 4. Preferably, the concentration of arginine is 0.6 to 1.2 M. Preferably, the concentration of the inorganic salt is 0.5 to 1 M. Preferably, the inorganic salt is selected from sodium chloride, potassium chloride, sodium citrate, and any mixture thereof, Preferably, the concentration of the buffer solution is 5 to 30 mM, more preferably 10 to 25 mM, Preferably, the buffer solution is selected from MES buffer solution, Tris buffer solution, acetic acid-sodium acetate buffer solution, and citric acid-sodium citrate buffer solution, Preferably, the nucleic acid lysate of the present invention further contains a pH adjuster, Preferably, the nucleic acid lysate further contains a metal chelating agent, Preferably, the concentration of the metal chelating agent is 1 to 20 mM, more preferably 5 to 10 mM, Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (for example, disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.), Preferably, the nucleic acid lysate consists of the above components, Preferably, the second solution is a buffer solution, and more preferably, the buffer solution is selected from DPBS buffer solution, Tris buffer solution, glycine-sodium hydroxide buffer solution, and disodium hydrogen phosphate-sodium hydroxide buffer solution. Preferably, the concentration of the Tris buffer solution, glycine-sodium hydroxide buffer solution, and disodium hydrogen phosphate-sodium hydroxide buffer solution is 1 to 30 mM, preferably 5 to 20 mM, Preferably, the third solution is a buffer solution, and more preferably, the buffer solution is selected from Tris buffer solution, MES buffer solution, acetic acid-sodium acetate buffer solution, and citric acid-sodium citrate buffer solution. Preferably, the concentration of the buffer solution is 1 to 20 mM, more preferably 5 to 15 mM. Preferably, the buffer solution further contains a metal chelating agent. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (for example, disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the concentration of the metal chelating agent is 0.5 to 10 mM, more preferably 1 to 5 mM, Preferably, the washing time of the nucleic acid extraction member is at least 20 s, preferably at least 30 s, more preferably 30 s to 2 min, Preferably, the time for nucleic acid release is at least 2 min, preferably at least 3 min, more preferably 3 to 5 min. The method according to claim 2 or 3, characterized in that.

5. A kit or set for nucleic acid extraction or detection, comprising the nucleic acid extraction member according to claim 1, Preferably, the kit or set further includes a first solution with a pH of 2 to 6, preferably 3 to 4, contained in a first container. More preferably, the kit or set further includes a second solution with a pH of 8 to 12, preferably 10 to 11, contained in a second container. Even more preferably, the kit or set further includes a third solution with a pH of 3 to 6, preferably 5 to 6, contained in a third container. Preferably, the first solution is a buffer solution. More preferably, the buffer solution is selected from MES buffer solution, Tris buffer solution, acetic acid-sodium acetate buffer solution, and citric acid-sodium citrate buffer solution. Preferably, the concentration of the buffer solution is 5 to 30 mM, more preferably 10 to 25 mM. Preferably, the first solution is a nucleic acid lysate. Preferably, the nucleic acid lysate contains 0.5 to 1.5 M arginine, 0.2 to 1.2 M inorganic salt, and a buffer solution, and has a pH of 2 to 6, preferably 3 to 4. Preferably, the concentration of arginine is 0.6 to 1.2 M. Preferably, the concentration of the inorganic salt is 0.5 to 1 M. Preferably, the inorganic salt is selected from sodium chloride, potassium chloride, sodium citrate, and any mixture thereof. Preferably, the concentration of the buffer solution is 5 to 30 mM, more preferably 10 to 25 mM. Preferably, the buffer solution is selected from MES buffer solution, Tris buffer solution, acetic acid-sodium acetate buffer solution, and citric acid-sodium citrate buffer solution. Preferably, the nucleic acid lysate of the present invention further contains a pH adjuster. Preferably, the nucleic acid lysate further contains a metal chelating agent. Preferably, the concentration of the metal chelating agent is 1 to 20 mM, preferably 5 to 10 mM. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (for example, disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the nucleic acid lysate consists of the above components. Preferably, the second solution is a buffer solution. More preferably, the buffer solution is selected from DPBS buffer solution, Tris buffer solution, glycine-sodium hydroxide buffer solution, and disodium hydrogen phosphate-sodium hydroxide buffer solution. Preferably, the concentration of Tris buffer solution, glycine-sodium hydroxide buffer solution, and disodium hydrogen phosphate-sodium hydroxide buffer solution is 1 to 30 mM, preferably 5 to 20 mM. Preferably, the third solution is a buffer solution. More preferably, the buffer solution is selected from Tris buffer solution, MES buffer solution, acetic acid-sodium acetate buffer solution, and citric acid-sodium citrate buffer solution. Preferably, the concentration of the buffer solution is 1 to 20 mM, more preferably 5 to 15 mM. Preferably, the buffer solution further contains a metal chelating agent. Preferably, the metal chelating agent is EDTA and / or a salt of EDTA (for example, disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.). Preferably, the concentration of the metal chelating agent is 0.5 to 10 mM, more preferably 1 to 5 mM. A kit or set for nucleic acid extraction or detection, characterized in that.

6. The nucleic acid extraction member is disposed at one end of the rod-shaped support member or is wrapped. Preferably, the length of the nucleic acid extraction member in the direction along the rod-shaped support member is 1 to 2 cm. Preferably, the other end of the rod-shaped support member is fixed inside the sealing members corresponding to the first container and the third container. Preferably, the first container and the third container are tubular, and the sealing member is a tube cap. Preferably, the rod-shaped support member has a breakable point at one end close to the nucleic acid extraction member. Preferably, the kit or set further includes a fourth container for packaging the nucleic acid extraction member. Preferably, the fourth container corresponds to the sealing member to which the rod-shaped support member is fixed. Preferably, the fourth container is tubular. The kit or set according to claim 5, characterized in that preferably, the fourth container does not contain other contents and is used only for packaging the nucleic acid extraction member.

7. The kit or set according to claim 5, characterized in that the nucleic acid extraction member is placed in the first solution.

8. The volume of the first solution is 1 to 10 mL, more preferably 1.5 to 3 mL. The volume of the second solution is 100 to 500 μL, more preferably 150 to 350 μL. The volume of the third solution is 1 to 10 mL, more preferably 2 to 4 mL. The kit or set according to any one of claims 5 to 7, characterized in that.

9. Use of polyacrylate, polyamide, polyester, polyurethane, or polylactic acid in nucleic acid extraction.

10. Use of polyacrylate, polyamide, polyester, polyurethane, or polylactic acid in the manufacture of a kit or set for nucleic acid extraction or nucleic acid detection, wherein. Preferably, the use is to manufacture the nucleic acid extraction member according to claim 1.

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