A device, kit and method for extracting nucleic acids

Abstract

This invention relates to the field of nucleic acid extraction, specifically to an apparatus, kit, and method for extracting and detecting nucleic acids. The apparatus, kit, and method can extract nucleic acids conveniently and efficiently, and significantly reduce the possibility of contamination during the nucleic acid extraction process.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction, specifically to an apparatus, reagent kit, and method for extracting nucleic acids. The apparatus, reagent kit, and method can extract nucleic acids conveniently and efficiently, and significantly reduce the possibility of contamination during the extraction process. Background Technology

[0002] Nucleic acid is the foundation of molecular biology research. Research in areas such as gene diagnosis, gene therapy, and identification of plant varieties using genes all require the extraction of high-quality nucleic acids. Currently, existing nucleic acid extraction and detection methods mainly have the following problems: (1) When the number of biological samples is huge, the processing of samples, nucleic acid extraction, and purification are prone to errors when done manually. Moreover, the overall operation steps are complex, making it impossible to extract nucleic acids efficiently and quickly. (2) Most molecular diagnostics need to be carried out in the laboratory. Many grassroots units do not have the conditions to establish a standard molecular diagnostic laboratory. In addition, the skill level of operators varies, and cross-contamination between samples is easy to occur during the nucleic acid extraction process. Even the laboratory is prone to contamination. (3) Most existing nucleic acid extraction and detection instruments are large in size and are not suitable for use at the sampling site. Furthermore, working in open outdoor environments makes it easier to cause sample contamination.

[0003] Therefore, there is an urgent need to develop a portable device for nucleic acid extraction that can reduce the impact of human operation and achieve fully enclosed operation, thereby reducing the possibility of nucleic acid extraction contamination and meeting the needs of rapid extraction and testing at the grassroots level or on-site. Summary of the Invention

[0004] To address the aforementioned problems, the inventors of this application, through extensive experimentation, have developed a device and reagent kit for extracting nucleic acids. This device and kit enable convenient and efficient nucleic acid extraction, significantly reducing the possibility of contamination during the extraction process and minimizing or even eliminating false positive results. Based on this, this application also provides a method for extracting nucleic acids using the aforementioned device or reagent kit. This method is simple and convenient, and can meet the needs of rapid extraction and testing at the grassroots level or on-site.

[0005] Therefore, in one aspect, this application provides an apparatus for extracting nucleic acids, comprising: a first container 101, a second container 102, and a gas storage device 103, wherein:

[0006] The first container 101 is used to contain or contain reagents for extracting nucleic acids; and has a first valve 106;

[0007] The first container 101 and the second container 102 are fluidly connected, and a second valve 107 is provided between them;

[0008] The second container 102 and the gas storage device 103 are in fluid communication; and

[0009] The second container 102 has an inlet 104 and an outlet 105. The inlet 104 is provided with a fourth valve 109, and the outlet 105 is provided with a fifth valve 110.

[0010] In some embodiments, the device is airtight. In some embodiments, the gas storage device 103 can contain or release gas. In some embodiments, the gas storage device 103 is variable in volume, such as a resilient or expandable container (e.g., an air bladder), or a container with a piston (e.g., a syringe). In some embodiments, a third valve 108 is provided between the second container 102 and the gas storage device 103.

[0011] The device described in this application minimizes or avoids gas exchange with the external environment by incorporating a gas storage device, thereby minimizing or avoiding the possibility of contamination during nucleic acid extraction. Without being limited by theory, the inventors have noted that experimental operations during nucleic acid extraction (e.g., shaking, agitation, etc.) can lead to aerosol generation, and gas exchange between these aerosols and the external environment (e.g., the experimental environment, or other nucleic acid samples extracted concurrently) can cause sample contamination. Therefore, minimizing or avoiding gas exchange between the sample and the external environment reduces or avoids the possibility of contamination during nucleic acid extraction. In some embodiments, the gas storage device 103 is an air bladder, which can draw in and expel gas through expansion and contraction, thereby reducing or avoiding gas exchange between the device and the external environment. In some embodiments, the gas storage device 103 is a container with a piston, which can draw in and expel gas through the reciprocating motion of the piston, thereby reducing or avoiding gas exchange between the device and the external environment.

[0012] In some embodiments, the first container 101 is tubular, with a first valve 106 at one end for connection to the drive device 111, and a second valve 107 at the other end for connection to the second container 102.

[0013] In some embodiments, the first container 101 contains a lysis buffer (e.g., cell lysis buffer), a washing buffer (e.g., nucleic acid washing buffer), and an elution buffer (e.g., nucleic acid elution buffer) for extracting nucleic acids.

[0014] In some embodiments, the lysis buffer, washing buffer, and elution buffer are separated from each other.

[0015] In some embodiments, the lysis buffer, washing buffer, and eluent are arranged in the first container 101 from proximal to distal end according to their distance from the second valve 107.

[0016] In some embodiments, the lysis buffer and the washing buffer are separated by a first spacer. In some embodiments, the washing buffer and the eluent are separated by a second spacer. In some embodiments, the first spacer and the second spacer are the same or different. In some embodiments, the first spacer and the second spacer are each independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin wax, silicone oil, and any combination thereof.

[0017] In some embodiments, the first container 101 contains a lysis buffer, a first spacer, a washing buffer, a second spacer, and an eluent, arranged sequentially from proximal to distal in the first container 101 according to their distance from the second valve 107.

[0018] In some embodiments, the washing liquid comprises one or more components (e.g., one, two, three, or more components), and the components are separated from each other by additional spacers. In some embodiments, the components have the same or different components. In some embodiments, the additional spacers are each independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin wax, silicone oil, and any combination thereof.

[0019] In some embodiments, a third spacer is provided between the eluent and the first valve 106. In some embodiments, the third spacer is selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin wax, silicone oil, and any combination thereof.

[0020] In such embodiments, the second container may be pre-placed with a magnet capable of adsorbing nucleic acids (e.g., magnetic beads). Alternatively, the magnet capable of adsorbing nucleic acids (e.g., magnetic beads) may be added to the second container after the device is activated.

[0021] Alternatively, in some embodiments, the first container 101 contains a washing solution (e.g., nucleic acid washing solution) and an elution solution (e.g., nucleic acid elution solution) for extracting nucleic acids.

[0022] In some embodiments, the washing solution and the elution solution are separated from each other.

[0023] In some embodiments, the washing liquid and the eluent are arranged in the first container 101 from the proximal end to the distal end according to their distance from the second valve 107.

[0024] In some embodiments, the washing solution and the eluent are separated by a first spacer.

[0025] In some embodiments, the first container 101 contains washing liquid, a first spacer, and an eluent, which are arranged in the first container 101 in order of distance from the second valve 107 from the proximal end to the distal end.

[0026] In some embodiments, the washing liquid comprises one or more components (e.g., one, two, three, or more components), and the components are separated from each other by additional spacers. In some embodiments, the components have the same or different components. In some embodiments, the additional spacers are each independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin wax, silicone oil, and any combination thereof.

[0027] In some embodiments, a second spacer is provided between the eluent and the first valve 106. In some embodiments, the first spacer and the second spacer may be the same or different. In some embodiments, the first spacer and the second spacer are each independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin wax, silicone oil, and any combination thereof.

[0028] In some embodiments, the second container may be pre-filled with a magnet capable of adsorbing nucleic acids (e.g., magnetic beads). Alternatively, the magnet capable of adsorbing nucleic acids (e.g., magnetic beads) may be added to the second container after the device is activated. Furthermore, in some embodiments, the second container may be pre-filled with a lysis buffer (e.g., cell lysis buffer) for nucleic acid extraction. Alternatively, the lysis buffer (e.g., cell lysis buffer) may be added to the second container after the device is activated. Therefore, in some embodiments, the second container 102 contains a lysis buffer (e.g., cell lysis buffer) for nucleic acid extraction, and / or a magnet capable of adsorbing nucleic acids (e.g., magnetic beads).

[0029] In some embodiments, one or more spacers may be applied between each pair of reagents, for example, one, two, three or more spacers. Each of the one or more spacers is independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin wax, silicone oil, and any combination thereof.

[0030] As used herein, the spacer used in this application can be a liquid, a gas, or a solid, and it is immiscible with the reagents used for nucleic acid extraction. In some embodiments, the spacer used in this application is stable and can separate the reagents at both ends even under conditions such as vibration, preventing the two reagents from contacting each other. Furthermore, it is understood that any substance capable of achieving this function can be used as the spacer in this application. Therefore, the spacer used in this application is not limited to the substances listed above.

[0031] In some embodiments, the first container 101 has a first chamber, a second chamber, and a third chamber separated from each other; wherein the first chamber is used to contain or contain a lysis buffer (e.g., cell lysis buffer) for extracting nucleic acids; the second chamber is used to contain or contain a washing buffer (e.g., nucleic acid washing buffer) for extracting nucleic acids; and the third chamber is used to contain or contain an elution buffer (e.g., nucleic acid elution buffer) for extracting nucleic acids; and the second valve 107 is capable of controlling the type of reagent flowing into the second container 102. In some embodiments, the second container may be pre-placed with a magnet capable of adsorbing nucleic acids (e.g., magnetic beads). Alternatively, the magnet capable of adsorbing nucleic acids (e.g., magnetic beads) may be added to the second container after the device is activated.

[0032] In some embodiments, the first container 101 has a first chamber and a second chamber separated from each other; wherein the first chamber is used to contain or contain a washing solution (e.g., nucleic acid washing solution) for extracting nucleic acids; the second chamber is used to contain or contain an elution solution (e.g., nucleic acid elution solution) for extracting nucleic acids; and the second valve 107 is capable of controlling the type of reagent flowing into the second container 102. In some embodiments, the second container may be pre-placed with a magnet capable of adsorbing nucleic acids (e.g., magnetic beads). Alternatively, the magnet capable of adsorbing nucleic acids (e.g., magnetic beads) may be added to the second container after the device is activated. Furthermore, in some embodiments, the second container 102 may be pre-placed with a lysis buffer (e.g., cell lysis buffer) for extracting nucleic acids. Alternatively, the lysis buffer (e.g., cell lysis buffer) may be added to the second container after the device is activated. Therefore, in some embodiments, the second container 102 contains a lysis buffer (e.g., cell lysis buffer) for extracting nucleic acids, and / or a magnet capable of adsorbing nucleic acids (e.g., magnetic beads).

[0033] In some embodiments, the first valve 106 is used to control the fluid communication between the first container 101 and the drive device 111. In some embodiments, the device further includes a drive device 111, which is fluidly connected to the first container 101 via the first valve 106. In some embodiments, the drive device 111 is detachable; in some embodiments, the drive device 111 is a drive pump.

[0034] In some embodiments, the second container 102 contains a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids, and / or a lysis buffer (e.g., cell lysis buffer) for extracting nucleic acids. In some embodiments, the magnetic beads are selected from one or more of the following: silica gel membrane magnetic beads, amino magnetic beads, hydroxyl magnetic beads, aldehyde magnetic beads, and cellulose-coated magnetic beads.

[0035] In some embodiments, the sample outlet 105 is connected to a waste tank 112, or a collection tank 113, or both, via a fifth valve 110. In some embodiments, the fifth valve 110 is capable of controlling the flow direction of fluid passing through it. In some embodiments, the collection tank 113 contains reagents for detecting nucleic acids. In some embodiments, the waste tank 112 and / or the collection tank 113 are removable.

[0036] In some embodiments, the nucleic acid is selected from DNA, RNA, or any combination thereof. The apparatus of this application can be used to extract DNA, RNA, or any combination thereof.

[0037] In a second aspect, this application provides a kit comprising the apparatus as described above. In some embodiments, the kit further comprises one or more components selected from the following: reagents for extracting nucleic acids, spacers, magnets (e.g., magnetic beads) capable of adsorbing nucleic acids, a drive device, a waste tank, and a collection tank.

[0038] In some embodiments, the kit further comprises reagents for extracting nucleic acids. In some embodiments, the reagents for extracting nucleic acids are selected from lysis buffers (e.g., cell lysis buffers), washing buffers (e.g., nucleic acid washing buffers), elution buffers (e.g., nucleic acid elution buffers), or any combination thereof. In some embodiments, the lysis buffer, washing buffer, and elution buffer are separated from each other.

[0039] In some embodiments, the kit further comprises one or more spacers. In some embodiments, each of the one or more spacers is independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

[0040] In some embodiments, the kit further comprises a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids. In some embodiments, the magnetic beads are selected from one or more of the following: silica gel membrane magnetic beads, amino magnetic beads, hydroxyl magnetic beads, aldehyde magnetic beads, and cellulose-coated magnetic beads.

[0041] In some embodiments, the kit further includes a drive device. In some embodiments, the drive device is a drive pump. In some embodiments, the drive device is fluidly connected to the first container 101 via a first valve 106.

[0042] In some embodiments, the kit further includes a waste liquid tank. In some embodiments, the waste liquid tank is fluidly connected to the sample outlet 105 via a fifth valve 110.

[0043] In some embodiments, the kit further includes a collection pool. In some embodiments, the collection pool is fluidly connected to the sample outlet 105 via a fifth valve 110. In some embodiments, the collection pool contains reagents for detecting nucleic acids.

[0044] In a third aspect, this application provides a method for extracting nucleic acids, the method comprising extracting nucleic acids from a sample using the apparatus or kit of this application.

[0045] In some embodiments, the method includes:

[0046] (1) Provide a sample containing nucleic acid, and the aforementioned device or the aforementioned kit;

[0047] (2) The sample is loaded into the second container 102 through the injection port 104 and the fourth valve 109 is closed; wherein the second container 102 contains a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids;

[0048] (3) The lysis buffer, a magnet capable of adsorbing nucleic acids (e.g., magnetic beads) are brought into contact with the sample in the second container 102, mixed, and incubated for an appropriate time;

[0049] (4) After incubation, open the third valve 108 and the fifth valve 110, and discharge all the solution in the second container 102 to the waste liquid pool 112 through the fifth valve 110, while retaining the magnet;

[0050] (5) Close the fifth valve 110, and use the driving device 111 to drive the washing liquid pre-loaded in the first container 101 into the second container 102, so that it can contact, mix and incubate with the magnet for a suitable time; wherein, during the driving process, the first valve 106, the second valve 107 and the third valve 108 are in the open state, and the fourth valve 109 and the fifth valve 110 are in the closed state; after the driving is completed, close the first valve 106 and the second valve 107, and optionally close the valve 108.

[0051] (6) After incubation, open the third valve 108 and the fifth valve 110, and discharge all the solution in the second container 102 to the waste liquid pool 112 through the fifth valve 110, while retaining the magnet;

[0052] Optionally, steps (5) and (6) may be repeated once or more;

[0053] (7) Close the fifth valve 110, and use the drive device 111 to drive the eluent pre-loaded in the first container 101 into the second container 102, where it contacts the magnet, mixes, and is incubated for a suitable time; wherein, during the driving process, the first valve 106, the second valve 107, and the third valve 108 are in the open state, and the fourth valve 109 and the fifth valve 110 are in the closed state; after the driving is completed, close the first valve 106 and the second valve 107, and optionally close valve 108;

[0054] (8) After incubation, open the third valve 108 and the fifth valve 110, and collect all the solution in the second container 102 into the collection pool 113 through the fifth valve 110, thereby obtaining a solution containing the nucleic acid to be extracted.

[0055] In some embodiments, the magnet used in step (2) has been pre-loaded into the second container 102. In some embodiments, the magnet used in step (2) is loaded into the second container 102 together with the sample. In some embodiments, the magnet used in step (2) is loaded into the second container 102 after the sample is loaded.

[0056] In some embodiments, the lysis buffer used in step (3) is preloaded into the second container 102. In some embodiments, the lysis buffer used in step (3) is loaded into the second container 102 together with the sample. In some embodiments, the lysis buffer used in step (3) is loaded into the second container 102 after the sample is loaded. In some embodiments, the lysis buffer used in step (3) is preloaded into the first container 101 and driven into the second container 102 by the drive device 111, wherein, during the drive, the first valve 106, the second valve 107, and the third valve 108 are in the open state, and the fourth valve 109 and the fifth valve 110 are in the closed state; after the drive is completed, the first valve 106 and the second valve 107 are closed, and optionally valve 108 is closed.

[0057] In some embodiments, the nucleic acids adsorbed on the magnet can be washed multiple times. Therefore, steps (5) and (6) can be repeated once or multiple times. In some embodiments, the first container 101 is tubular and contains one or more sections of washing solution, thereby allowing the nucleic acids adsorbed on the magnet to be washed once or multiple times.

[0058] In some embodiments, the extracted nucleic acid is detected after step (8). In some embodiments, the collection pool 113 contains reagents for detecting the nucleic acid. In some embodiments, the extracted nucleic acid is detected in the collection pool 113.

[0059] In a fourth aspect, this application provides the use of the apparatus or kit described above for extracting nucleic acids.

[0060] Terminology Definition

[0061] As used herein, the term "fluid" refers to a substance that can flow and deform under shear forces. For example, the most common fluids are gases and liquids. As used herein, the term "fluid connection" refers to a connection between two components in a device that allows fluids (e.g., gases and liquids) to flow between the two components.

[0062] As used herein, the term "reagent for nucleic acid extraction" refers to the reagents used in the nucleic acid extraction process. Reagents for nucleic acid extraction typically include lysis buffers, nucleic acid washing buffers, and nucleic acid elution buffers.

[0063] As used herein, the term "lysis buffer" refers to a solution in which nucleic acids in a sample are released. Such lysis buffers are well known to those skilled in the art and are readily prepared or commercially available. In some embodiments, the lysis buffer is capable of lysing cells, releasing nucleic acids contained within the cells (e.g., genomic DNA, plasmid DNA, mitochondrial DNA, chloroplast DNA, total RNA, mRNA, tRNA, miRNA, etc.) and dissolving / releasing them in the lysis buffer. In such embodiments, the lysis buffer is also referred to as a cell lysis buffer.

[0064] As used herein, the term "nucleic acid washing solution" refers to a solution used during nucleic acid extraction to remove impurities (e.g., cell debris, proteins, polysaccharides, plasma membranes, etc.) from a system containing nucleic acids. Such nucleic acid washing solutions are well known to those skilled in the art and are readily prepared or commercially available. In this document, "nucleic acid washing solution" and "washing solution" are used interchangeably.

[0065] As used herein, the term "nucleic acid eluent" refers to a solution used in nucleic acid extraction to separate nucleic acids from a system containing nucleic acids, which can dissolve and stably store the nucleic acids. Such nucleic acid eluents are well known to those skilled in the art and are readily prepared or commercially available. In this document, "nucleic acid eluent" and "elution" are used interchangeably.

[0066] Those skilled in the art will understand that the composition of the lysis buffer (e.g., cell lysis buffer), nucleic acid washing buffer, and nucleic acid elution buffer can be adjusted according to factors such as the type of sample from which nucleic acids are to be extracted (e.g., cell culture, isolated tissue, body fluid, etc.), the type of nucleic acid to be extracted (e.g., DNA, such as genomic DNA, plasmid DNA, mitochondrial DNA, chloroplast DNA, etc.; or RNA, such as total RNA, mRNA, tRNA, miRNA, etc.), and the extraction method used. Furthermore, such adjustments are entirely within the capabilities of those skilled in the art.

[0067] For example, when the nucleic acid to be extracted is plasmid DNA from cell culture, the lysis buffer typically contains cell lysis reagents (e.g., SDS, Triton X-100, NP-40, guanidine isothiocyanate (GITC), etc.) and salts (e.g., Tris, EDTA, NaCl, etc.). When the nucleic acid to be extracted is genomic DNA, the lysis buffer may also contain hexadecyltrimethylammonium bromide (CTAB). For example, a commonly used cell lysis buffer formulation for DNA extraction is 4M guanidine hydrochloride, 50mM Tris-HCl, 10mM EDTA, 15% Triton X100, pH 6.5. Commonly used washing solutions for DNA washing may include salts (e.g., Tris, EDTA, NaCl, etc.) and ethanol. For example, a commonly used washing solution formulation for DNA washing is 100mM NaCl, 50mM Tris-HCl (pH 7.8), 75% anhydrous ethanol. Commonly used elution solutions for DNA elution include TE buffer or sterile water. TE buffer is typically prepared from Tris and EDTA. For example, a common elution solution for DNA elution is 10 mM Tris-HCl, 1 mM EDTA, pH 8.5.

[0068] For example, when the nucleic acid to be extracted is total RNA from cells, the lysis buffer typically contains cell-lysing agents (e.g., Trizol), RNase inhibitors (e.g., 8-hydroxyquinoline, β-mercaptoethanol, DEPC, etc.), and protein denaturing agents (e.g., GIT, GuHCl, etc.). Commonly used washing buffers for RNA washing may include RNase inhibitors (e.g., 8-hydroxyquinoline, β-mercaptoethanol, DEPC, etc.) and ethanol. Commonly used elution buffers for eluting RNA may be RNase-free sterile water.

[0069] It is readily understood that the lysis buffer, nucleic acid washing buffer, and nucleic acid elution buffer involved in this invention are not limited to the formulations and components listed above, nor are they limited to the formulations and components used in the examples. Furthermore, as mentioned above, they can be adjusted and varied according to actual needs.

[0070] As used herein, the term "magnet capable of adsorbing nucleic acids" refers to a surface-modified magnetic material (e.g., magnetic particles) that specifically recognizes and binds to nucleic acid molecules at a microscopic interface. In some embodiments, the magnetic material is paramagnetic (e.g., paramagnetic particles), and preferably superparamagnetic (e.g., superparamagnetic particles). Paramagnetic magnets are particularly advantageous because they can rapidly aggregate in a magnetic field and disperse uniformly upon leaving the field. Methods for adsorbing / extracting nucleic acids using magnetic materials (also known as "magnetic bead methods") are well known in the art, and various magnetic beads for nucleic acid extraction have been developed, including, for example, DNA extraction magnetic beads and RNA extraction magnetic beads, such as silica gel membrane magnetic beads, amino magnetic beads, hydroxyl magnetic beads, aldehyde magnetic beads, cellulose-coated magnetic beads, etc. Such magnetic beads are commercially available. In some cases, using magnetic bead methods to extract nucleic acids is particularly advantageous because the method can be easily automated. Therefore, in some preferred embodiments, the apparatus and kit of the present invention can be used for nucleic acid extraction via magnetic bead methods.

[0071] As used herein, the term "nucleic acid" includes single-stranded and double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In this application, nucleic acids include, but are not limited to, DNA, such as plasmid DNA, genomic DNA, mitochondrial DNA, chloroplast DNA, etc.; and RNA, such as total RNA, mRNA, tRNA, miRNA, etc.

[0072] Beneficial effects of the invention

[0073] The nucleic acid extraction device, reagent kit, and method provided in this application enable the extraction and detection of nucleic acids from samples under sealed conditions, and greatly reduce or avoid gas exchange between the device / sample and the external environment. This significantly reduces the possibility of environmental contamination and cross-contamination between samples during nucleic acid extraction, and reduces or even eliminates false positive results. Furthermore, the nucleic acid extraction device of this application has a simple structure, is easy to carry, and the extraction method is easy to operate, making it suitable for the needs of rapid extraction and detection at grassroots or on-site locations. Attached Figure Description

[0074] Figure 1 This application illustrates an apparatus for extracting nucleic acids, comprising: a first container 101, a second container 102, and a gas storage device 103, wherein:

[0075] The first container 101 contains a lysis buffer, a washing buffer, and an eluent separated from each other by a spacer; and is connected to the drive device 111 via a first valve 106.

[0076] The first container 101 and the second container 102 are fluidly connected, and a second valve 107 is provided between them;

[0077] The second container 102 and the gas storage device 103 are fluidly connected, and a third valve 108 is provided between them;

[0078] The second container 102 has an inlet 104 and an outlet 105, and the inlet 104 is provided with a fourth valve 109; the second container may contain magnetic beads;

[0079] The sample outlet 105 is provided with a fifth valve 110; and the sample outlet 105 is connected to the waste liquid tank 112 or the collection tank 113 via the fifth valve 110.

[0080] Figure 2 This application illustrates an apparatus for extracting nucleic acids, comprising: a first container 201, a second container 202, and a gas storage device 203, wherein:

[0081] The first container 201 contains washing liquid and eluent separated from each other by a spacer; and is connected to the drive device 211 via the first valve 206;

[0082] The first container 201 and the second container 202 are fluidly connected, and a second valve 207 is provided between them;

[0083] The second container 202 and the gas storage device 203 are fluidly connected, and a third valve 208 is provided between them;

[0084] The second container 202 has an inlet 204 and an outlet 205, and the inlet 204 is provided with a fourth valve 209; the second container may contain lysis buffer and / or magnetic beads;

[0085] The sample outlet 205 is equipped with a fifth valve 210; and the sample outlet 205 is connected to the waste liquid tank 212 or the collection tank 213 via the fifth valve 210.

[0086] Figure 3 This application illustrates an apparatus for extracting nucleic acids, comprising: a first container 301, a second container 302, and a gas storage device 303, wherein:

[0087] The first container 301 has a first chamber, a second chamber, and a third chamber that are separated from each other; wherein the first chamber contains a lysis buffer, the second chamber contains a washing buffer, and the third chamber contains an elution buffer;

[0088] The first container is connected to the drive device 311 via the first valve 306;

[0089] The first container 301 and the second container 302 are fluidly connected, and a second valve 307 is provided between them; the second valve 307 can control the type of fluid flowing into the second container 302.

[0090] The second container 302 and the gas storage device 303 are fluidly connected, and a third valve 308 is provided between them;

[0091] The second container 302 has an inlet 304 and an outlet 305, and the inlet 304 is provided with a fourth valve 309; the second container may contain magnetic beads;

[0092] The sample outlet 305 is provided with a fifth valve 310; and the sample outlet 305 is connected to the waste liquid tank 312 or the collection tank 313 via the fifth valve 310.

[0093] Figure 4 This application illustrates an apparatus for extracting nucleic acids, comprising: a first container 401, a second container 402, and a gas storage device 403, wherein:

[0094] The first container 401 has a first chamber and a second chamber separated from each other; wherein the first chamber contains washing liquid and the second chamber contains eluent;

[0095] The first container is connected to the drive device 411 via the first valve 406;

[0096] The first container 401 and the second container 402 are fluidly connected, and a second valve 407 is provided between them; the second valve 407 can control the type of fluid flowing into the second container 402.

[0097] The second container 402 and the gas storage device 403 are fluidly connected, and a third valve 408 is provided between them;

[0098] The second container 402 has an inlet 404 and an outlet 405, and the inlet 404 is provided with a fourth valve 409; the second container may contain magnetic beads and / or lysis buffer;

[0099] The sample outlet 405 is provided with a fifth valve 410; and the sample outlet 405 is connected to the waste liquid tank 412 or the collection tank 413 via the fifth valve 410. Detailed Implementation

[0100] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0101] Example 1: Device Structure and Usage

[0102] Figure 1 This application discloses an apparatus for extracting nucleic acids, comprising: a first container 101, a second container 102, and a gas storage device 103. The first container 101, arranged from proximal to distal end according to its distance from a second valve 107, contains lysis buffer, a first spacer, a washing buffer, a second spacer, and an elution buffer; and is connected to a drive device 111 via a first valve 106. The first container 101 and the second container 102 are fluidly connected, and a second valve 107 is disposed between them. The second container 102 and the gas storage device 103 are fluidly connected, and a third valve 108 is disposed between them. The second container 102 has an inlet 104 and an outlet 105, the inlet 104 being equipped with a fourth valve 109. The second container may contain magnetic beads. The outlet 105 is equipped with a fifth valve 110; and the outlet 105 is connected to a waste liquid tank 112 or a collection tank 113 via the fifth valve 110.

[0103] When using this device to extract nucleic acids from a sample, firstly, the sample containing nucleic acids is loaded into a second container 102 containing a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids through the inlet 104, and the fourth valve 109 is closed. Then, the lysis buffer in the first container 101 is driven into the second container 102 by the drive device 111, wherein during the drive, the first valve 106, the second valve 107, and the third valve 108 are open, and the fourth valve 109 and the fifth valve 110 are closed. During this period, the gas in the second container 102 enters the gas storage device 103 through the third valve 108, instead of entering the external environment. After the drive is completed, the first valve 106 and the second valve 107 are closed, and optionally valve 108 is closed, allowing the lysis buffer, the magnet, and the sample to mix in the second container 102 and incubate for a suitable time. After incubation, the third valve 108 and the fifth valve 110 are opened, and all the solution in the second container 102 is discharged to the waste liquid pool 112 through the fifth valve 110, while the magnet is retained. During this period, the gas in the gas storage device 103 will enter the second container 102 through the third valve 108 to prevent the entry of gas from the external environment.

[0104] Subsequently, the fifth valve 110 is closed, and the washing liquid in the first container 101 is driven into the second container 102 by the driving device 111, allowing it to contact, mix, and incubate with the magnet for a suitable time. During this process, the first valve 106, the second valve 107, and the third valve 108 are open, while the fourth valve 109 and the fifth valve 110 are closed. During this period, the gas in the second container 102 will enter the gas storage device 103 through the third valve 108, instead of entering the external environment. After the driving process is completed, the first valve 106 and the second valve 107 are closed, and valve 108 is optionally closed, allowing the washing liquid, magnet, and sample to mix and incubate in the second container 102 for a suitable time. After incubation, the third valve 108 and the fifth valve 110 are opened, and all the solution in the second container 102 is discharged to the waste liquid pool 112 through the fifth valve 110, while the magnet is retained. During this period, the gas in the gas storage device 103 will enter the second container 102 through the third valve 108, preventing the entry of gases from the external environment. Optionally, the washing process can be repeated once or multiple times.

[0105] Subsequently, the fifth valve 110 is closed, and the eluent in the first container 101 is driven into the second container 102 using the drive device 111, where it comes into contact with the magnet, mixes, and is incubated for a suitable time. During this process, the first valve 106, the second valve 107, and the third valve 108 are open, while the fourth valve 109 and the fifth valve 110 are closed. During this period, the gas in the second container 102 enters the gas storage device 103 through the third valve 108, instead of entering the external environment. After the drive is completed, the first valve 106 and the second valve 107 are closed, and optionally valve 108 is closed, allowing the eluent, magnet, and sample to mix and incubate in the second container 102 for a suitable time. After incubation, the third valve 108 and the fifth valve 110 are opened, and all the solution in the second container 102 is discharged into the collection tank 113 through the fifth valve 110, thereby obtaining a solution containing the nucleic acid to be extracted. During this period, the gas in the gas storage device 103 will enter the second container 102 through the third valve 108 to prevent the entry of gas from the external environment.

[0106] In this device, the reservoir 103 buffers air, allowing for the intake and exhaust of gases. The presence of the reservoir enables repeated gas circulation within the entire device. Therefore, the entire device does not exchange gases with the outside, achieving a sealed design and preventing sample and environmental contamination during nucleic acid extraction. The second container 102 is used for lysis, binding, washing, and elution during nucleic acid extraction. The sample outlet 105 is used to discharge waste liquid generated during nucleic acid extraction (connected to waste liquid tank 112) or collect extracted nucleic acids (connected to collection tank 113). The waste liquid tank 112 and collection tank 113 can be detachable, or the flow direction of the fluids (waste liquid and nucleic acid solution) can be controlled via a fifth valve 110.

[0107] Figure 2 This application presents another apparatus for extracting nucleic acids, comprising: a first container 201, a second container 202, and a gas storage device 203, wherein: the first container 201 contains washing solution, a first spacer, and an elution solution sequentially from proximal to distal end according to its distance from the second valve 207; and is connected to a drive device 211 via the first valve 206; the first container 201 and the second container 202 are fluidly connected, and the second valve 207 is disposed between them; the second container 202 and the gas storage device 203 are fluidly connected, and a third valve 208 is disposed between them; the second container 202 has an inlet 204 and an outlet 205, the inlet 204 being provided with a fourth valve 209; the second container may contain lysis buffer and / or magnetic beads; the outlet 205 is provided with a fifth valve 210; and the outlet 205 is connected to a waste liquid tank 212 or a collection tank 213 via the fifth valve 210.

[0108] Figure 2 The device shown is Figure 1 The main difference in the apparatus shown is that the reagents pre-loaded in the first container are different. Figure 2 In this device, the first container 201 contains only washing solution, a first spacer, and eluent, but not lysis buffer. Therefore, during use, the drive device 211 is no longer used to drive the lysis buffer into the second container 202; instead, the lysis buffer is pre-loaded into the second container 202, or added to the second container 202 through the injection port 204. Furthermore, Figure 2 The method of using the device shown is the same as Figure 1 The apparatus shown is essentially the same.

[0109] Figure 3This application illustrates another apparatus for extracting nucleic acids, comprising: a first container 301, a second container 302, and a gas storage device 303. The first container 301 has a first chamber, a second chamber, and a third chamber separated from each other. The first chamber contains a lysis buffer, the second chamber contains a washing buffer, and the third chamber contains an elution buffer. The first container is connected to a drive device 311 via a first valve 306. The first container 301 and the second container 302 are fluidly connected, and a second valve 306 is provided between them. 7. The second valve 307 can control the type of fluid flowing into the second container 302; the second container 302 and the gas storage device 303 are fluidly connected, and a third valve 308 is provided between them; the second container 302 has an inlet 304 and an outlet 305, and the inlet 304 is provided with a fourth valve 309; the second container may contain magnetic beads; the outlet 305 is provided with a fifth valve 310; and the outlet 305 is connected to a waste liquid tank 312 or a collection tank 313 via the fifth valve 310.

[0110] Figure 3 The device shown is Figure 1 The main difference in the devices shown lies in the design of the first container. Figure 3 In this apparatus, the first container 301 separates the lysis buffer, washing buffer, and eluent through different chambers (rather than through a spacer), and the type of reagent flowing into the second container 302 is controlled by a second valve 307 (rather than by the order of reagents). Therefore, during the use of this apparatus, the position of the second valve 307 needs to be adjusted according to the specific steps required to control the type of reagent flowing into the second container 302. In addition, Figure 3 The method of using the device shown is the same as Figure 1 The apparatus shown is essentially the same.

[0111] Figure 4This application discloses an apparatus for extracting nucleic acids, comprising: a first container 401, a second container 402, and a gas storage device 403. The first container 401 has a first chamber and a second chamber separated from each other; the first chamber contains a washing solution, and the second chamber contains an elution solution. The first container is connected to a drive device 411 via a first valve 406. The first container 401 and the second container 402 are fluidly connected, and a second valve 407 is provided between them. The type of fluid flowing into the second container 402 can be controlled; the second container 402 and the gas storage device 403 are fluidly connected, and a third valve 408 is provided between them; the second container 402 has an inlet 404 and an outlet 405, and the inlet 404 is provided with a fourth valve 409; the second container may contain magnetic beads and / or lysis buffer; the outlet 405 is provided with a fifth valve 410; and the outlet 405 is connected to a waste liquid tank 412 or a collection tank 413 via the fifth valve 410.

[0112] Figure 4 The device shown is Figure 3 The main difference in the apparatus shown is that the reagents pre-loaded in the first container are different. Figure 4 In this device, the first container 401 contains only washing solution, a first spacer, and eluent, but not lysis buffer. Therefore, during use, the drive device 411 is no longer used to drive the lysis buffer into the second container 402; instead, the lysis buffer is pre-loaded into the second container 402, or added to the second container 402 through the injection port 404. Furthermore, Figure 4 The method of using the device shown is the same as Figure 3 The apparatus shown is essentially the same.

[0113] Figure 1-4 The drive unit, waste liquid tank and collection tank used can be included in the device or can be detachable and provided separately as independent accessories.

[0114] Example 2: Nucleic Acid Extraction and Detection

[0115] In this embodiment, using Figure 1The apparatus designed for nucleic acid extraction comprises a first tubular container with an inner diameter of 1 mm. The first container contains 50 μL of eluent (formulated as 10 mM Tris-HCl, 1 mM EDTA, pH 8.5), 3 ml of washing buffer (formulated as 100 mM NaCl, 50 mM Tris-HCl (pH 7.8), 75% anhydrous ethanol), and 2 ml of lysis buffer (formulated as 4 M guanidine hydrochloride, 50 mM Tris-HCl, 10 mM EDTA, 15% Triton X100, pH 6.5). Both the first and second spacers are air. The second container is pre-loaded with silica gel magnetic beads (containing iron tetroxide and silica) for nucleic acid extraction. The reservoir is an air bag. The driving mechanism is a pump. Before use, all valves are closed to prevent leakage of reagents from the first container and to prevent external gases from entering the apparatus.

[0116] The specific steps for extracting nucleic acids using the above-mentioned device are as follows:

[0117] (1) Open the fourth valve and add 200 μL of nucleic acid sample; then close the fourth valve.

[0118] (2) Open the first valve, the second valve, and the third valve, and drive the pyrolysis solution in the first container into the second container by driving the pump. Then, close the first valve, the second valve, and the third valve.

[0119] (3) Use a shaker or magnetic stirrer to thoroughly mix the sample, magnetic beads, and lysis buffer in the second container; incubate at room temperature for 2-5 minutes to complete sample lysis and nucleic acid binding to the magnetic beads. After binding is complete, turn off the instrument or magnetic stirrer.

[0120] (4) Use a magnet to attract the magnetic beads in the second container to the inner wall of the second container.

[0121] (5) Open the third and fifth valves to discharge the waste pyrolysis liquid in the second container into the waste liquid pool through the sample outlet. Then, close the fifth valve.

[0122] (6) Open the first valve, the second valve, and the third valve, and drive the pump to drive the washing liquid in the first container into the second container. Then, close the first valve, the second valve, and the third valve.

[0123] (7) Use a shaker or magnetic stirrer to thoroughly mix the sample, magnetic beads, and washing solution in the second container; incubate at room temperature for 1-2 minutes to wash the nucleic acids bound to the magnetic beads. After washing, turn off the instrument or magnetic stirrer.

[0124] (8) Use a magnet to attract the magnetic beads in the second container to the inner wall of the second container.

[0125] (9) Open the third and fifth valves to discharge the waste washing liquid in the second container into the waste liquid pool through the sample outlet. Then, close the fifth valve.

[0126] (10) Open the first valve, the second valve, and the third valve, and drive the eluent in the first container into the second container by driving the pump. Then, close the first valve, the second valve, and the third valve.

[0127] (11) Use a shaker or a magnetic stirrer to thoroughly mix the sample, magnetic beads, and elution buffer in the second container; incubate at room temperature for 2-5 minutes to elute the nucleic acids bound to the magnetic beads. After elution, turn off the instrument or magnetic stirrer.

[0128] (12) Use a magnet to attract the magnetic beads in the second container to the inner wall of the second container.

[0129] (13) Open the third and fifth valves to discharge the nucleic acid elution buffer from the second container into the collection tank through the sample outlet. The collection tank contains pre-loaded lyophilized reagents for nucleic acid detection (including 50 μmol primers, 50 μmol probes, 5 U Taq DNA polymerase, 10×PCR buffer, Mg... 2+ 100 mmol, dNTP 10 mmol). Then, close the third and fifth valves.

[0130] (14) The nucleic acids in the collection pool are automatically detected using the accompanying instruments. These instruments are capable of PCR amplification and real-time fluorescence detection; in this embodiment, a real-time fluorescence PCR instrument is used. The thermal cycling module provides the collection pool with different reaction temperatures required for PCR amplification, and the fluorescence signal detection module detects the fluorescence signal of the PCR amplification reactants at specific stages of the reaction.

[0131] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An apparatus for extracting nucleic acids, comprising: The first container (101), the second container (102), and the gas storage device (103) are, wherein: The tubular first container (101) is used to contain or contain reagents for extracting nucleic acids; and has a first valve (106). The first container (101) and the second container (102) are fluidly connected, and a second valve (107) is provided between them. The second container (102) and the gas storage device (103) are fluidly connected; and The second container (102) has an inlet (104) and an outlet (105), the inlet (104) being provided with a fourth valve (109); and the outlet (105) being provided with a fifth valve (110). The device is airtight, and the gas storage device (103) is of variable volume and can hold or release gas; Furthermore, a third valve (108) is provided between the second container (102) and the gas storage device (103).

2. The apparatus of claim 1, wherein, The gas storage device (103) is a flexible or stretchable container, or a container with a piston.

3. The apparatus of claim 1, wherein, The gas storage device (103) is an air bag or a syringe.

4. The apparatus according to claim 1 or 2, wherein, The first container (101) has a first valve (106) at one end for connecting to the drive device (111) and a second valve (107) at the other end for connecting to the second container (102).

5. The apparatus of claim 4, wherein, The first container (101) has the following features (3-1) or (3-2): (3-1) The first container (101) contains a lysis buffer, a washing buffer, and an elution buffer for extracting nucleic acids; or, (3-2) The first container (101) contains a washing solution and an elution solution for extracting nucleic acids.

6. The apparatus of claim 5, wherein, The lysis buffer, washing buffer, and elution buffer are separated from each other.

7. The apparatus of claim 6, wherein, The first container (101) contains lysis buffer, first spacer, washing buffer, second spacer and eluent, and they are arranged in the first container (101) in order of distance from the second valve (107) from the proximal end to the distal end.

8. The apparatus of claim 7, wherein, The first spacer and the second spacer are each independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, and any combination thereof.

9. The apparatus of claim 5, wherein, The washing solution and the elution solution are separated from each other.

10. The apparatus of claim 9, wherein, The first container (101) contains washing liquid, a first spacer and an eluent, which are arranged in the first container (101) from proximal end to distal end according to their distance from the second valve (107).

11. The apparatus of claim 10, wherein, The first spacer and the second spacer are each independently selected from air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, and any combination thereof.

12. The apparatus of claim 10, wherein, The second container (102) contains a lysis buffer for extracting nucleic acids.

13. The apparatus of claim 5, wherein it has one or more features selected from the following: (1) The lysis buffer used for nucleic acid extraction is a cell lysis buffer; (2) The washing solution is a nucleic acid washing solution; (3) The elution buffer is a nucleic acid elution buffer.

14. The apparatus of claim 4, wherein, The device has one or more of the following technical features: (1) The first valve (106) is used to control the fluid connection between the first container (101) and the drive device (111); (2) The second container (102) contains a magnet capable of adsorbing nucleic acids, and / or a lysis buffer for extracting nucleic acids; (3) The sample outlet (105) is connected to the waste liquid tank (112) or the collection tank (113) via the fifth valve (110), or the waste liquid tank (112) and the collection tank (113). (4) The nucleic acid is selected from DNA, RNA or any combination thereof.

15. The apparatus of claim 1, further comprising a drive (111) in fluid communication with the first container (101) via a first valve (106).

16. A kit comprising the apparatus according to any one of claims 1-15.

17. A method for extracting nucleic acids, the method comprising extracting nucleic acids from a sample using the apparatus of any one of claims 1-15 or the kit of claim 16.

18. A method for extracting nucleic acids, the method comprising: (1) Provide a sample containing nucleic acid, and the apparatus of any one of claims 1-15 or the kit of claim 16; (2) The sample is loaded into the second container (102) through the injection port (104) and the fourth valve (109) is closed; wherein the second container (102) contains a magnet capable of adsorbing nucleic acids; (3) The lysis buffer, the magnet that can adsorb nucleic acid, and the sample are brought into contact, mixed, and incubated for an appropriate time in the second container (102); (4) After incubation, open the third valve (108) and the fifth valve (110), and discharge all the solution in the second container (102) to the waste liquid pool (112) through the fifth valve (110), while retaining the magnet; (5) Close the fifth valve (110) and use the driving device (111) to drive the washing liquid pre-loaded in the first container (101) into the second container (102) to contact, mix and incubate with the magnet for a suitable time; wherein, during the driving process, the first valve (106), the second valve (107) and the third valve (108) are in the open state, and the fourth valve (109) and the fifth valve (110) are in the closed state; after the driving is completed, close the first valve (106) and the second valve (107), and optionally close the third valve (108); (6) After incubation, open the third valve (108) and the fifth valve (110), and discharge all the solution in the second container (102) to the waste liquid pool (112) through the fifth valve (110), while retaining the magnet; (7) Close the fifth valve (110), and use the driving device (111) to drive the eluent pre-loaded in the first container (101) into the second container (102), so that it can contact the magnet, mix and incubate for a suitable time; wherein, during the driving process, the first valve (106), the second valve (107) and the third valve (108) are in the open state, and the fourth valve (109) and the fifth valve (110) are in the closed state; after the driving is completed, close the first valve (106) and the second valve (107), and optionally close the third valve (108); (8) After incubation, open the third valve (108) and the fifth valve (110) to collect all the solutions in the second container (102) into the collection pool (113) through the fifth valve (110) to obtain a solution containing the nucleic acid to be extracted.

19. The method of claim 18, wherein the lysis buffer has been pre-loaded into the second container (102), or the lysis buffer is loaded into the second container (102) together with the sample, or the lysis buffer is loaded into the second container (102) after the sample is loaded; or the lysis buffer has been pre-loaded into the first container (101) and driven into the second container (102) by the driving device (111), wherein, During the driving process, the first valve (106), the second valve (107) and the third valve (108) are in the open state, and the fourth valve (109) and the fifth valve (110) are in the closed state; after the driving is completed, the first valve (106) and the second valve (107) are closed, and the third valve (108) is optionally closed.

20. The method of claim 19, wherein steps (5) and (6) are repeated once or more.

21. Use of the apparatus according to any one of claims 1-15 or the kit according to claim 16 for extracting nucleic acids.

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