CN215668051U

CN215668051U - Device for extracting nucleic acid

Info

Publication number: CN215668051U
Application number: CN202120072323.7U
Authority: CN
Inventors: 占伟; 杨毅; 唐宇; 曹甜甜
Original assignee: XIAMEN ZEESAN BIOTECH CO Ltd
Current assignee: XIAMEN ZEESAN BIOTECH CO Ltd
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2022-01-28
Anticipated expiration: 2031-01-12

Abstract

The invention relates to the field of nucleic acid extraction, in particular to a device, a kit and a method for extracting and detecting nucleic acid. The present application provides an apparatus for extracting nucleic acid, comprising: first container, second container and gas storage device, wherein: the first container is used for containing or containing a reagent for extracting nucleic acid; and, having a first valve; the first container and the second container are in fluid communication, and a second valve is disposed therebetween; the second container is in fluid communication with the gas storage means; the second container is provided with a sample inlet and a sample outlet, and the sample inlet is provided with a fourth valve; and the sample outlet is provided with a fifth valve. The device, the kit and the method can conveniently and efficiently extract nucleic acid, and obviously reduce the possibility of pollution in the process of extracting nucleic acid.

Description

Device for extracting nucleic acid

Technical Field

The invention relates to the field of nucleic acid extraction, in particular to a device, a kit and a method for extracting nucleic acid, which can conveniently and efficiently extract nucleic acid and obviously reduce the possibility of pollution in the process of extracting nucleic acid.

Background

Nucleic acid is the basis of molecular biology research, and high-quality nucleic acid extraction is required for research in aspects of gene diagnosis, gene therapy, plant variety identification by using genes and the like. At present, the existing nucleic acid extraction and detection means mainly have the following problems: (1) under the condition of huge quantity of biological samples, errors are easy to occur in the manual operation of sample treatment, nucleic acid extraction and purification, and the overall operation steps are complex, so that the nucleic acid extraction cannot be efficiently and quickly carried out; (2) most molecular diagnosis needs to be carried out in a laboratory, a plurality of basic units do not have the condition for establishing a standard molecular diagnosis laboratory, and due to different proficiency of operators, cross contamination among samples can easily occur in the process of extracting nucleic acid, and even the laboratory can easily cause contamination; (3) most of the existing instruments for extracting and detecting nucleic acid have larger volume, are not suitable for being used in a sampling field, and are easier to cause sample pollution when working in an outdoor open environment.

Therefore, it is urgently needed to develop a device for nucleic acid extraction, which is portable, can reduce the influence of manual operation, and can realize fully-closed operation, thereby reducing the possibility of nucleic acid extraction pollution, and meeting the requirements of basic level or on-site rapid extraction and detection.

Disclosure of Invention

In order to solve the above problems, the inventors of the present application have made extensive experiments to develop a device and a kit for extracting nucleic acid, which can conveniently and efficiently extract nucleic acid, and can significantly reduce the possibility of contamination during the extraction of nucleic acid, and reduce or even eliminate false positive detection results. Based on the above, the application also provides a method for extracting nucleic acid by using the device or the kit, and the method is simple and convenient and can meet the requirements of basic layer or on-site rapid extraction and detection.

Accordingly, in one aspect, the present application provides an apparatus for extracting nucleic acids comprising: first container 101, second container 102, and gas storage device 103, wherein:

the first container 101 is used for containing or containing a reagent for extracting nucleic acid; and, having a first valve 106;

the first container 101 and the second container 102 are in fluid communication, and a second valve 107 is disposed therebetween;

the second container 102 is in fluid communication with the gas storage means 103; and is

The second container 102 has a sample inlet 104 and a sample outlet 105, and the sample inlet 104 is provided with a fourth valve 109; the sample outlet 105 is provided with a fifth valve 110.

In certain embodiments, the device is air tight. In some embodiments, the gas storage device 103 can contain or exclude gas. In some embodiments, the gas reservoir 103 is variable in volume, such as a container with elasticity or stretchability (e.g., a balloon), or a container with a plunger (e.g., a syringe). In some embodiments, a third valve 108 is disposed between the second container 102 and the gas storage device 103.

The device of this application comes minimize or avoids the gas exchange with external environment through setting up gas storage device to minimize or avoid the possibility of polluting among the nucleic acid extraction process. Without being bound by theory, the present inventors have noted that experimental manipulations (e.g., shaking, etc.) during nucleic acid extraction can lead to aerosol generation, and that gas exchange between the aerosol and the external environment (e.g., the experimental environment, or other nucleic acid samples extracted in parallel) can lead to contamination of the sample. Thus, minimizing or avoiding gas exchange between the sample and the external environment reduces or avoids the possibility of contamination during the nucleic acid extraction process. In some embodiments, the gas storage device 103 is a balloon that can draw in and expel gas by inflation and deflation of the balloon, thereby reducing or avoiding gas exchange between the device of the present application and the external environment. In some embodiments, the gas storage device 103 is a container with a piston, which can suck and discharge gas by the reciprocating motion of the piston, thereby reducing or avoiding the gas exchange between the device and the external environment.

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

In certain embodiments, the first container 101 has the following feature (3-1) or (3-2):

(3-1) the first container (101) contains a lysis solution (e.g., a cell lysis solution) for extracting nucleic acid, a washing solution (e.g., a nucleic acid washing solution) and an elution solution (e.g., a nucleic acid elution solution);

alternatively, the first and second electrodes may be,

(3-2) the first container (101) contains a washing solution (e.g., a nucleic acid washing solution) for extracting nucleic acid and an elution solution (e.g., a nucleic acid elution solution).

In certain embodiments, the lysing solution, wash solution, and eluent are separated from one another.

In some embodiments, the lysing solution, wash solution, and elution solution are arranged in the first container 101 at a distance from the second valve 107 from the proximal end to the distal end.

In certain embodiments, the lysis solution and wash solution are separated by a first spacer. In certain embodiments, the wash solution and the eluent are separated by a second spacer. In certain embodiments, the first and second spacing agents are the same or different. In certain embodiments, the first and second spacing agents are each independently selected from the group consisting of air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

In some embodiments, the first container 101 contains a lysis solution, a first spacer, a wash solution, a second spacer and an elution solution, and they are arranged in the first container 101 in order from the proximal end to the distal end at a distance from the second valve 107.

In certain embodiments, the wash solution comprises one or more portions (e.g., 1, 2, 3, or more portions), and each portion is separated from each other by an additional spacing agent. In certain embodiments, the various moieties have the same or different compositions. In certain embodiments, each of the additional spacing agents is independently selected from the group consisting of air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

In some embodiments, a third spacer is disposed between the eluent and the first valve 106. In certain embodiments, the third spacer is selected from the group consisting of air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

In such embodiments, the second container may be pre-loaded with a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids. Alternatively, a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids can also be added to the second container after activation of the device.

In certain embodiments, the wash solution and the elution solution are separated from each other.

In some embodiments, the wash solution and the elution solution are arranged in the first container 101 from a proximal end to a distal end at a distance from the second valve 107.

In certain embodiments, the wash solution and the eluent are separated by a first spacer.

In some embodiments, the first container 101 contains a wash solution, a first spacer and an eluent, and they are arranged in the first container 101 in order from proximal to distal, in a distance from the second valve 107.

In some embodiments, a second spacer is disposed between the eluent and the first valve 106. In certain embodiments, the first and second spacing agents are the same or different. In certain embodiments, the first and second spacing agents are each independently selected from the group consisting of air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

In certain embodiments, the second container may be pre-loaded with a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids. Alternatively, a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids can also be added to the second container after activation of the device. Further, in certain embodiments, the second container may be pre-loaded with a lysis solution (e.g., a cell lysis solution) for extracting nucleic acids. Alternatively, a lysis solution for extracting nucleic acids (e.g., a cell lysate) may be added to the second container after activation of the device. Thus, in certain embodiments, the second container 102 contains a lysis solution (e.g., a cell lysis solution) for extracting nucleic acids, and/or a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids.

In certain embodiments, one or more spacing agents, e.g., 1, 2, 3 or more spacing agents, may be applied between each two reagents. Each of the one or more spacing agents is independently selected from the group consisting of air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

As used herein, a spacer as used herein may be a liquid, may be a gas, or may be a solid, and is immiscible with reagents used to extract nucleic acids. In some embodiments, the spacer used in the present application has stable properties, and can separate the reagents at both ends thereof even under conditions such as vibration, so that the two reagents do not contact each other. Further, it is to be understood that any substance capable of achieving such a function may be used as the spacer in the present application. Therefore, the spacer used in the present application is not limited to the above-listed substances.

In certain embodiments, the first container 101 has a first chamber, a second chamber, and a third chamber that are separated from one another; wherein the first chamber is adapted to contain or contain a lysis solution (e.g. a cell lysis solution) for extracting nucleic acid; a second chamber for containing or containing a wash solution for extracting nucleic acids (e.g., a nucleic acid wash solution); a third chamber for containing or containing an eluent for extracting nucleic acids (e.g., a nucleic acid eluent); also, the second valve 107 can control the type of reagent flowing into the second container 102. In certain embodiments, the second container may be pre-loaded with a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids. Alternatively, a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids can also be added to the second container after activation of the device.

In certain embodiments, the first container 101 has a first chamber and a second chamber separated from each other; wherein the first chamber is for containing or containing a wash solution for extracting nucleic acids (e.g., a nucleic acid wash solution); a second chamber for containing or containing an eluate for extracting nucleic acids (e.g., a nucleic acid eluate); also, the second valve 107 can control the type of reagent flowing into the second container 102. In certain embodiments, the second container may be pre-loaded with a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids. Alternatively, a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids can also be added to the second container after activation of the device. Furthermore, in certain embodiments, the second container 102 may be pre-loaded with a lysis solution (e.g., a cell lysis solution) for extracting nucleic acids. Alternatively, a lysis solution for extracting nucleic acids (e.g., a cell lysate) may be added to the second container after activation of the device. Thus, in certain embodiments, the second container 102 contains a lysis solution (e.g., a cell lysis solution) for extracting nucleic acids, and/or a magnet (e.g., magnetic beads) capable of adsorbing nucleic acids.

In certain embodiments, the first valve 106 is used to control the fluid communication of the first container 101 with the actuating device 111. In certain embodiments, the apparatus also has an actuation device 111 in fluid communication with the first container 101 through the first valve 106. In certain embodiments, the drive device 111 is removable; in certain embodiments, the drive device 111 is a drive pump.

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

In some embodiments, the sample outlet 105 is connected to the waste liquid pool 112, or the collecting pool 113, or both the waste liquid pool 112 and the collecting pool 113 via the fifth valve 110. In some embodiments, the fifth valve 110 can control the direction of fluid flow through the fifth valve 110. In certain embodiments, the collection reservoir 113 comprises reagents for detecting nucleic acids. In certain embodiments, the waste reservoir 112 and/or the collection reservoir 113 are removable.

In certain embodiments, the nucleic acid is selected from DNA, RNA, or any combination thereof. The device of the present application may be used to extract DNA, RNA or any combination thereof.

In a second aspect, the present application provides a kit comprising a device as described above. In certain embodiments, the kit further comprises one or more components or components selected from the group consisting of: a reagent for extracting nucleic acid, a spacer, a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acid, a driving device, a waste reservoir, and a collection reservoir.

In certain embodiments, the kit further comprises reagents for extracting nucleic acids. In certain embodiments, the reagent for extracting nucleic acids is selected from a lysate (e.g., a cell lysate), a wash solution (e.g., a nucleic acid wash solution), an eluate (e.g., a nucleic acid eluate), or any combination thereof. In certain embodiments, the lysing solution, wash solution, and eluent are separated from one another.

In certain embodiments, the kit further comprises one or more spacing agents. In certain embodiments, the one or more spacing agents are each independently selected from the group consisting of air, mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, and any combination thereof.

In certain embodiments, the kit further comprises a magnet (e.g., a magnetic bead) capable of adsorbing the nucleic acid. In certain embodiments, the magnetic beads are selected from one or more of the following: silica gel plasma membrane magnetic beads, amino magnetic beads, hydroxyl magnetic beads, aldehyde magnetic beads and cellulose coated magnetic beads.

In certain embodiments, the kit further comprises a drive device. In certain embodiments, the drive device is a drive pump. In certain embodiments, the actuating device can be in fluid communication with the first container 101 through the first valve 106.

In certain embodiments, the kit further comprises a waste reservoir. In some embodiments, the waste reservoir can be in fluid communication with the outlet 105 through a fifth valve 110.

In certain embodiments, the kit further comprises a collection reservoir. In some embodiments, the collection reservoir can be in fluid communication with the outlet 105 through a fifth valve 110. In certain embodiments, the collection reservoir comprises reagents for detecting nucleic acids.

In a third aspect, the present application provides a method of extracting nucleic acid, the method comprising extracting nucleic acid from a sample using a device of the present application or a kit of the present application.

In certain embodiments, the method comprises:

(1) providing a sample comprising nucleic acids, and, the aforementioned device or the aforementioned kit;

(2) loading the sample into the second container 102 through the sample inlet 104 and closing the fourth valve 109; wherein the second container 102 contains a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids;

(3) contacting the lysate, a magnet capable of adsorbing nucleic acids (e.g., magnetic beads), and the sample in the second container 102, mixing and incubating for a suitable time;

(4) after incubation, the third valve 108 and the fifth valve 110 are opened, and all the solution in the second container 102 is drained through the fifth valve 110 to the waste reservoir 112, leaving the magnet;

(5) closing the fifth valve 110, driving the washing solution pre-loaded in the first container 101 into the second container 102 by the driving device 111, contacting with the magnet, mixing and incubating for a suitable time; wherein, during the driving, the first valve 106, the second valve 107 and the third valve 108 are in an open state, and the fourth valve 109 and the fifth valve 110 are in a closed state; after the driving is finished, closing the first valve 106 and the second valve 107, and optionally closing the valve 108;

(6) after incubation, the third valve 108 and the fifth valve 110 are opened, and all the solution in the second container 102 is drained through the fifth valve 110 to the waste reservoir 112, leaving the magnet;

optionally, repeating steps (5) and (6) one or more times;

(7) closing the fifth valve 110, driving the eluent pre-loaded in the first container 101 into the second container 102 by the driving device 111, contacting with the magnet, mixing and incubating for a suitable time; wherein, during the driving, the first valve 106, the second valve 107 and the third valve 108 are in an open state, and the fourth valve 109 and the fifth valve 110 are in a closed state; after the driving is finished, closing the first valve 106 and the second valve 107, and optionally closing the valve 108;

(8) after incubation, the third valve 108 and the fifth valve 110 are opened, and all the solution in the second container 102 is collected into the collection tank 113 through the fifth valve 110, thereby obtaining a solution containing the nucleic acid to be extracted.

In certain embodiments, the magnet used in step (2) has been pre-loaded in the second container 102. In certain embodiments, the magnet used in step (2) is loaded into the second container 102 along with the sample. In certain embodiments, the magnet used in step (2) is loaded into the second container 102 after sample loading.

In certain embodiments, the lysis solution used in step (3) has been pre-loaded in second vessel 102. In certain embodiments, the lysis solution used in step (3) is loaded into the second container 102 along with the sample. In certain embodiments, the sample used in step (3) is loaded into the second container 102 after loading. In certain embodiments, the lysis solution used in step (3) has been pre-loaded in the first container 101 and driven into the second container 102 by the driving device 111, wherein during driving, the first valve 106, the second valve 107 and the third valve 108 are in an open state, and the fourth valve 109 and the fifth valve 110 are in a closed state; after the actuation is finished, the first valve 106 and the second valve 107 are closed, and optionally the valve 108 is closed.

In certain embodiments, the nucleic acid adsorbed on the magnet can be washed multiple times. Thus, steps (5) and (6) may be repeated one or more times. In certain embodiments, the first container 101 is tubular and contains one or more sections of wash solution, whereby one or more washes of nucleic acids adsorbed on the magnet can be performed.

In certain embodiments, after performing step (8), the extracted nucleic acid is detected. In certain embodiments, the collection reservoir 113 comprises reagents for detecting nucleic acids. In certain embodiments, the extracted nucleic acids are detected in collection reservoir 113.

In a fourth aspect, the present application provides the use of a device as described above or a kit as described above for extracting nucleic acids.

Definition of terms

As used herein, the term "fluid" refers to a flowable substance that deforms under shear forces. For example, the most common fluids are gases and liquids. As used herein, the term "fluid communication" is used to refer to a connection relationship between two components in a device that enables fluid (e.g., gas and liquid) to be communicated between the two components.

As used herein, the term "reagent for extracting nucleic acid" refers to a reagent used in a nucleic acid extraction process. The reagent for extracting nucleic acid generally comprises lysis solution, nucleic acid washing solution and nucleic acid eluent.

As used herein, the term "lysate" refers to a solution in which nucleic acids in a sample are freed. Such lysates are well known to those skilled in the art and may be conveniently formulated or commercially available. In certain embodiments, the lysate is capable of lysing the cells, releasing nucleic acids (e.g., genomic DNA, plasmid DNA, mitochondrial DNA, chloroplast DNA, total RNA, mRNA, tRNA, miRNA, etc.) contained in the cells, and dissolving/dissociating in the lysate. In such embodiments, the lysate is also referred to as a cell lysate.

As used herein, the term "nucleic acid wash solution" refers to a solution used to remove impurities (e.g., cell debris, proteins, polysaccharides, plasma membranes, etc.) from a system comprising nucleic acids during nucleic acid extraction. Such nucleic acid washing solutions are well known to those skilled in the art and may be conveniently formulated or commercially available. Herein, "nucleic acid wash solution" and "wash solution" are used interchangeably.

As used herein, the term "nucleic acid eluent" refers to a solution for separating nucleic acids from a system containing nucleic acids during nucleic acid extraction, which is capable of dissolving nucleic acids and stably storing nucleic acids. Such nucleic acid eluents are well known to those skilled in the art and may be conveniently formulated or commercially available. Herein, "nucleic acid eluent" and "eluent" are used interchangeably.

It will be appreciated by those skilled in the art that the composition of the lysis solution (e.g., cell lysate), nucleic acid wash solution and nucleic acid eluate can be adjusted depending on the type of sample from which nucleic acids are to be extracted (e.g., cell culture, ex vivo tissue, body fluids, etc.), the type of nucleic acids 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.), the extraction method used, etc. And such adjustments are well within the ability of those skilled in the art.

For example, when the nucleic acid to be extracted is plasmid DNA in cell culture, the lysate may typically contain reagents to lyse cells (e.g., SDS, Triton X-100, NP-40, Guanidinium Isothiocyanate (GITC), etc.) and salts (e.g., Tris, EDTA, NaCl, etc.). When the nucleic acid to be extracted is genomic DNA, the lysate may also typically comprise cetyltrimethylammonium bromide (CTAB). For example, a commonly used formulation of cell lysate for DNA extraction may be 4M guanidine hydrochloride, 50mM Tris-HCl, 10mM EDTA, 15% Triton X100, pH 6.5. Commonly used washing solutions for washing DNA may include salts (e.g., Tris, EDTA, NaCl, etc.) and ethanol, among others. For example, a commonly used washing solution formulation for washing DNA may be 100mM NaCl, 50mM Tris-HCl (pH 7.8), 75% absolute ethanol. The elution solution commonly used for eluting DNA may be TE solution or sterile water, wherein the TE solution is usually prepared by Tris and EDTA. For example, a commonly used formulation of eluent for elution of DNA may be 10mM Tris-HCl, 1mM EDTA, pH 8.5.

For example, when the nucleic acid to be extracted is total RNA in a cell, the lysis solution may typically contain an agent that lyses the cell (e.g., Trizol, etc.), an rnase inhibitor (e.g., 8-hydroxyquinoline, β -mercaptoethanol, DEPC, etc.), a protein denaturing agent (e.g., GIT, GuHCl, etc.). Commonly used washing solutions for washing RNA may include RNase inhibitors (e.g., 8-hydroxyquinoline, β -mercaptoethanol, DEPC, etc.) and ethanol, among others. A commonly used eluent for eluting RNA may be sterile water containing no RNase.

It is to be understood that the lysis solution, the nucleic acid washing solution and the nucleic acid eluting solution according to the present invention are not limited to the above-listed formulations and components, and are not limited to the formulations and components used in the examples, and may be adjusted and changed according to actual needs, as described above.

As used herein, the term "magnet capable of adsorbing nucleic acids" refers to a surface-modified and modified magnetic substance (e.g., magnetic particles) that is capable of specifically recognizing and binding to nucleic acid molecules at a microscopic interface. In certain embodiments, the magnetic substance is a paramagnetic substance (e.g., a paramagnetic particle), and preferably a superparamagnetic substance (e.g., a superparamagnetic particle). Magnets with paramagnetism can be concentrated rapidly in the magnetic field and can be dispersed uniformly after leaving the magnetic field, and are therefore particularly advantageous. Methods of adsorbing/extracting nucleic acids using magnetic substances (also referred to as "magnetic bead method") are well known in the art, and various magnetic beads for extracting nucleic acids have been developed, including, for example, DNA extraction magnetic beads and RNA extraction magnetic beads, such as silica membrane magnetic beads, amino magnetic beads, hydroxyl magnetic beads, aldehyde magnetic beads, cellulose-coated magnetic beads, and the like. Such magnetic beads are commercially available. In some cases, the use of magnetic bead method for nucleic acid extraction is particularly advantageous, because the method can be easily automated. Thus, in certain preferred embodiments, the devices and kits of the present invention can be used to extract nucleic acids by magnetic bead methods.

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

Advantageous effects of the invention

The nucleic acid extraction device, the kit and the method can realize extraction and detection from the sample to the nucleic acid in a sealed state, and greatly reduce or avoid gas exchange between the device and the sample and the external environment, thereby remarkably reducing the possibility of environmental pollution and cross pollution among the samples in the nucleic acid extraction process, and reducing or even avoiding false positive detection results. In addition, the nucleic acid extraction device of the application has the advantages of simple structure, portability, easy operation of the extraction method, and capability of adapting to the requirements of basic-level or on-site rapid extraction and detection.

Drawings

FIG. 1 shows a device designed for extracting nucleic acids according to the present application, comprising: first container 101, second container 102, and gas storage device 103, wherein:

the first container 101 contains a lysis solution, a washing solution and an elution solution separated from each other by a spacer; and is connected to the driving device 111 via the first valve 106;

the second container 102 and the gas storage device 103 are in fluid communication, and a third valve 108 is disposed therebetween;

the second container 102 has a sample inlet 104 and a sample outlet 105, and the sample inlet 104 is provided with a fourth valve 109; the second container may contain magnetic beads;

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

FIG. 2 shows a device designed for nucleic acid extraction according to the present application, comprising: a first container 201, a second container 202, and a gas storage device 203, wherein:

the first container 201 contains a wash solution and an eluent separated from each other by a spacer; and, via the first valve 206, to the driving means 211;

the first container 201 and the second container 202 are in fluid communication, and a second valve 207 is disposed therebetween;

the second container 202 is in fluid communication with the gas reservoir 203, and a third valve 208 is disposed therebetween;

the second container 202 is provided with a sample inlet 204 and a sample outlet 205, and the sample inlet 204 is provided with a fourth valve 209; the second container can contain lysis solution and/or magnetic beads;

the sample outlet 205 is provided with a fifth valve 210; the sample outlet 205 is connected to a waste liquid tank 212 or a collection tank 213 via a fifth valve 210.

FIG. 3 shows a device designed for extracting nucleic acids according to the present application, comprising: a first container 301, a second container 302, and a gas storage device 303, wherein:

the first container 301 has a first chamber, a second chamber and a third chamber separated from each other; wherein the first chamber contains a lysis solution, the second chamber contains a washing solution, and the third chamber contains an eluent;

the first container is connected with the driving device 311 through a first valve 306;

the first container 301 and the second container 302 are in fluid communication, and a second valve 307 is disposed therebetween; the second valve 307 is capable of controlling the type of fluid flowing into the second container 302;

the second container 302 is in fluid communication with the gas reservoir 303, and a third valve 308 is disposed therebetween;

the second container 302 is provided with a sample inlet 304 and a sample outlet 305, and the sample inlet 304 is provided with a fourth valve 309; the second container may contain magnetic beads;

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

FIG. 4 shows an apparatus for extracting nucleic acid designed in the present application, comprising: a first container 401, a second container 402, and a gas storage device 403, wherein:

the first container 401 has a first chamber and a second chamber separated from each other; wherein the first chamber contains a wash solution and the second chamber contains an eluent;

the first container is connected with a driving device 411 through a first valve 406;

the first container 401 and the second container 402 are in fluid communication, and a second valve 407 is disposed therebetween; the second valve 407 is capable of controlling the type of fluid flowing into the second container 402;

the second container 402 is in fluid communication with the gas reservoir 403, and a third valve 408 is disposed therebetween;

the second container 402 has a sample inlet 404 and a sample outlet 405, and the sample inlet 404 is provided with a fourth valve 409; the second container can contain magnetic beads and/or lysis solution;

the sample outlet 405 is provided with a fifth valve 410; the sample outlet 405 is connected to a waste liquid tank 412 or a collection tank 413 via a fifth valve 410.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: device construction and method of use

FIG. 1 shows a device designed for extracting nucleic acids according to the present application, comprising: first container 101, second container 102, and gas storage device 103, wherein: the first container 101 sequentially comprises lysis solution, a first spacing agent, washing solution, a second spacing agent and eluent from the near end to the far end according to the distance from the second valve 107; and is connected to the driving device 111 via the first valve 106; the first container 101 and the second container 102 are in fluid communication, and a second valve 107 is disposed therebetween; the second container 102 and the gas storage device 103 are in fluid communication, and a third valve 108 is disposed therebetween; the second container 102 has a sample inlet 104 and a sample outlet 105, and the sample inlet 104 is provided with a fourth valve 109; the second container may contain magnetic beads; the sample outlet 105 is provided with a fifth valve 110; the sample outlet 105 is connected to a waste liquid tank 112 or a collection tank 113 via a fifth valve 110.

When nucleic acid is extracted from a sample using the apparatus, first, a sample containing nucleic acid is loaded into the second container 102 containing a magnet (e.g., magnetic bead) capable of adsorbing nucleic acid through the sample inlet 104, and the fourth valve 109 is closed. Then, the lysate in the first container 101 is driven into the second container 102 by the driving device 111, wherein during the driving, the first valve 106, the second valve 107 and the third valve 108 are in an open state, and the fourth valve 109 and the fifth valve 110 are in a closed state. During this time, the gas in the second container 102 will enter the gas storage device 103 through the third valve 108, rather than entering the external environment. After the actuation is finished, the first valve 106 and the second valve 107, and optionally the valve 108, are closed, and the lysate, magnet and sample are mixed well in the second container 102 and incubated 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 drained through the fifth valve 110 to the waste reservoir 112, leaving the magnet. During this period, the gas in the gas storage device 103 will enter the second container 102 through the third valve 108, and the gas from the external environment will be prevented from entering.

Subsequently, the fifth valve 110 is closed, and the washing solution in the first container 101 is driven into the second container 102 by the driving device 111, contacted with the magnet, mixed and incubated for a suitable time; wherein, during driving, the first valve 106, the second valve 107 and the third valve 108 are in an open state, and the fourth valve 109 and the fifth valve 110 are in a closed state. During this time, the gas in the second container 102 will enter the gas storage device 103 through the third valve 108, rather than entering the external environment. After the actuation is finished, the first valve 106 and the second valve 107, and optionally the valve 108, are closed, and the wash, magnet and sample are mixed in the second container 102 and incubated 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 drained through the fifth valve 110 to the waste reservoir 112, leaving the magnet. During this period, the gas in the gas storage device 103 will enter the second container 102 through the third valve 108, and the gas from the external environment will be prevented from entering. Optionally, the washing process can be repeated one or more times.

Subsequently, the fifth valve 110 is closed, and the eluent in the first container 101 is driven into the second container 102 by the driving device 111, contacted with the magnet, mixed and incubated for a suitable time; wherein, during driving, the first valve 106, the second valve 107 and the third valve 108 are in an open state, and the fourth valve 109 and the fifth valve 110 are in a closed state. During this time, the gas in the second container 102 will enter the gas storage device 103 through the third valve 108, rather than entering the external environment. After the actuation is complete, the first valve 106 and the second valve 107, and optionally the valve 108, are closed, and the eluate, magnet, and sample are mixed in the second container 102 and incubated 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 drained through the fifth valve 110 to the collection tank 113, 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, and the gas from the external environment will be prevented from entering.

In this device, the reservoir device 103 is used to buffer air, which can be inhaled and expelled. The presence of the reservoir means allows for repeated circulation of gas throughout the device. Therefore, the whole device does not exchange gas with the outside, the sealing of the device can be realized, and the pollution of samples and the environment in the nucleic acid extraction process can be avoided. The second vessel 102 is used to perform lysis, binding, washing and elution during nucleic acid extraction. The outlet 105 is used for discharging waste liquid generated in the nucleic acid extraction process (connected to the waste liquid tank 112) or collecting the extracted nucleic acid (connected to the collection tank 113). The waste liquid tank 112 and the collection tank 113 may be detachable, or the flow direction of the fluid (waste liquid and nucleic acid solution) may be controlled by the fifth valve 110.

FIG. 2 shows another apparatus designed for nucleic acid extraction according to the present application, comprising: a first container 201, a second container 202, and a gas storage device 203, wherein: the first container 201 comprises a washing solution, a first spacer and an eluent from the proximal end to the distal end in sequence according to the distance from the second valve 207; and, via the first valve 206, to the driving means 211; the first container 201 and the second container 202 are in fluid communication, and a second valve 207 is disposed therebetween; the second container 202 is in fluid communication with the gas reservoir 203, and a third valve 208 is disposed therebetween; the second container 202 is provided with a sample inlet 204 and a sample outlet 205, and the sample inlet 204 is provided with a fourth valve 209; the second container can contain lysis solution and/or magnetic beads; the sample outlet 205 is provided with a fifth valve 210; the sample outlet 205 is connected to a waste liquid tank 212 or a collection tank 213 via a fifth valve 210.

The device shown in fig. 2 differs from the device shown in fig. 1 primarily in that the reagent pre-loaded in the first container is different. In the device of fig. 2, the first container 201 is loaded with only the wash solution, the first spacer and the eluent, and does not contain the lysis solution. Thus, during use of the device, the drive means 211 is no longer used to drive the lysis solution into the second container 202, but the lysis solution is pre-loaded in the second container 202 or added to the second container 202 through the sample inlet 204. Otherwise, the method of use of the apparatus shown in fig. 2 is substantially the same as that of the apparatus shown in fig. 1.

FIG. 3 shows another apparatus designed for nucleic acid extraction according to the present application, comprising: a first container 301, a second container 302, and a gas storage device 303, wherein: the first container 301 has a first chamber, a second chamber and a third chamber separated from each other; wherein the first chamber contains a lysis solution, the second chamber contains a washing solution, and the third chamber contains an eluent; the first container is connected with the driving device 311 through a first valve 306; the first container 301 and the second container 302 are in fluid communication, and a second valve 307 is disposed therebetween, the second valve 307 being capable of controlling the type of fluid flowing into the second container 302; the second container 302 is in fluid communication with the gas reservoir 303, and a third valve 308 is disposed therebetween; the second container 302 is provided with a sample inlet 304 and a sample outlet 305, and the sample inlet 304 is provided with a fourth valve 309; the second container may contain magnetic beads; the sample outlet 305 is provided with a fifth valve 310; the sample outlet 305 is connected to a waste liquid tank 312 or a collection tank 313 via a fifth valve 310.

The device shown in fig. 3 differs from the device shown in fig. 1 primarily in the way in which the first container is designed. In the device of fig. 3, the first container 301 separates the lysate, wash solution and eluent by different chambers (rather than by spacers), and the second valve 307 (rather than by the order of the reagents) is used to control the type of reagents that flow into the second container 302. Thus, during use of the device, it is necessary to adjust the position of the second valve 307 as required by the particular procedure, thereby controlling the type of reagent flowing into the second container 302. Otherwise, the method of use of the apparatus shown in fig. 3 is substantially the same as that of the apparatus shown in fig. 1.

FIG. 4 shows an apparatus for extracting nucleic acid designed in the present application, comprising: a first container 401, a second container 402, and a gas storage device 403, wherein: the first container 401 has a first chamber and a second chamber separated from each other; wherein the first chamber contains a wash solution and the second chamber contains an eluent; the first container is connected with a driving device 411 through a first valve 406; the first container 401 and the second container 402 are in fluid communication, and a second valve 407 is disposed therebetween, the second valve 407 being capable of controlling the type of fluid flowing into the second container 402; the second container 402 is in fluid communication with the gas reservoir 403, and a third valve 408 is disposed therebetween; the second container 402 has a sample inlet 404 and a sample outlet 405, and the sample inlet 404 is provided with a fourth valve 409; the second container can contain magnetic beads and/or lysis solution; the sample outlet 405 is provided with a fifth valve 410; the sample outlet 405 is connected to a waste liquid tank 412 or a collection tank 413 via a fifth valve 410.

The device shown in fig. 4 differs from the device shown in fig. 3 primarily in that the reagent pre-loaded in the first container is different. In the device of fig. 4, the first container 401 is loaded with only the wash solution, the first spacer and the eluent, and does not contain the lysis solution. Thus, during use of the device, the drive means 411 is no longer used to drive the lysis fluid into the second container 402, but the lysis fluid is pre-loaded in the second container 402 or added to the second container 402 via the sample inlet 404. Otherwise, the method of use of the apparatus shown in fig. 4 is substantially the same as that of the apparatus shown in fig. 3.

The drive means, waste reservoir and collection reservoir employed in fig. 1-4 may be contained within the apparatus or may be removable and provided separately as separate kits.

Example 2: extraction and detection of nucleic acids

In this example, the nucleic acid was extracted using the apparatus designed in FIG. 1, wherein the first vessel was a tubular vessel having an inner wall diameter of 1 mm. The first container was filled with 50. mu.L of eluent (formulation 10mM Tris-HCl, 1mM EDTA, pH 8.5), 3ml of washing solution (formulation 100mM NaCl, 50mM Tris-HCl (pH 7.8), 75% absolute ethanol), and 2ml of lysis solution (formulation 4M guanidine hydrochloride, 50mM Tris-HCl, 10mM EDTA, 15% Triton X100, pH 6.5). The first and second spacers are both air. The second container is loaded with silica membrane magnetic beads (containing ferroferric oxide and silica) for extracting nucleic acid in advance. The reservoir means is a balloon. The driving device is a driving pump. Before the kit is not used, all valves are in a closed state, so that the reagent in the first container is prevented from flowing out, and gas in the external environment is prevented from entering the device.

The nucleic acid is extracted by using the device, and the specific steps are as follows:

(1) opening the fourth valve, and adding 200 μ L of nucleic acid sample; then, the fourth valve is closed.

(2) And opening the first valve, the second valve and the third valve, and driving the pump to drive the lysate in the first container to flow into the second container. Then, the first valve, the second valve and the third valve are closed.

(3) Fully and uniformly mixing the sample, the magnetic beads and the lysis solution in the second container by using a vibration instrument or a magnetic stirrer; incubation at room temperature for 2-5 minutes completed lysis of the sample and binding of nucleic acids to the magnetic beads. After the binding is complete, the instrument or magnetic stirrer is turned off.

(4) The magnetic beads in the second container are adsorbed to the inner wall of the second container by using a magnet.

(5) And opening the third valve and the fifth valve, and discharging the waste pyrolysis liquid in the second container into a waste liquid pool through the sample outlet. Then, the fifth valve is closed.

(6) And opening the first valve, the second valve and the third valve, and driving the pump to drive the washing liquid in the first container to flow into the second container. Then, the first valve, the second valve and the third valve are closed.

(7) Fully and uniformly mixing the sample, the magnetic beads and the washing solution in the second container by using a vibration instrument or a magnetic stirrer; the incubation was performed at room temperature for 1-2 minutes to complete the washing of the nucleic acids bound to the magnetic beads. After washing was complete, the instrument or magnetic stirrer was turned off.

(8) The magnetic beads in the second container are adsorbed to the inner wall of the second container by using a magnet.

(9) And opening the third valve and the fifth valve, and discharging the waste cleaning solution in the second container into the waste solution tank through the sample outlet. Then, the fifth valve is closed.

(10) The first valve, the second valve and the third valve are opened, and the eluent in the first container is driven to flow into the second container by driving the pump. Then, the first valve, the second valve and the third valve are closed.

(11) Fully and uniformly mixing the sample, the magnetic beads and the eluent in the second container by using a shaking instrument or a magnetic stirrer; the incubation was performed at room temperature for 2-5 minutes, thereby eluting the nucleic acid bound to the magnetic beads. After elution was complete, the instrument or magnetic stirrer was turned off.

(12) The magnetic beads in the second container are adsorbed to the inner wall of the second container by using a magnet.

(13) Opening the third valve and the fifth valve, discharging the nucleic acid eluate in the second container into a collection pool containing a pre-loaded reagent (including 50 μmol of primer, 50 μmol of probe, 5U of Taq DNA polymerase, 10 XPCR buffer, Mg) for detecting nucleic acid in the form of lyophilized powder²⁺100mmol, dNTP 10 mmol). Then, the third and fifth valves are closed.

(14) The nucleic acid in the collecting pool is automatically detected by a matched instrument. The instrument used in the present embodiment is an instrument capable of performing PCR amplification and real-time fluorescence detection, and the real-time fluorescence PCR instrument is used in the present embodiment. The thermal cycle module provides different reaction temperatures for the collecting pool according to the PCR amplification requirement, and the fluorescence signal detection module performs fluorescence signal detection on the PCR amplification reactant at a specific reaction stage.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present 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: a first container (101), a second container (102) and a gas storage means (103), wherein:

the first container (101) is used for containing or containing a reagent for extracting nucleic acid; and, having a first valve (106);

the first container (101) and the second container (102) are in fluid communication and a second valve (107) is disposed therebetween;

said second container (102) being in fluid communication with gas storage means (103); and is

The second container (102) is provided with a sample inlet (104) and a sample outlet (105), and the sample inlet (104) is provided with a fourth valve (109); the sample outlet (105) is provided with a fifth valve (110).

2. The apparatus for extracting nucleic acid according to claim 1, wherein the apparatus is airtight.

3. The apparatus for nucleic acid isolation according to claim 1, wherein the gas storage means (103) can contain or exclude gas.

4. The apparatus for extracting nucleic acid according to claim 1, wherein the gas storage means (103) is variable in volume.

5. The apparatus for nucleic acid isolation according to claim 1, wherein the gas storage means (103) is a container having elasticity or elasticity, or a container with a plunger.

6. The apparatus for extracting nucleic acid according to claim 5, wherein the container having elasticity or stretchability is a balloon.

7. The apparatus for extracting nucleic acid according to claim 5, wherein the container with the plunger is a syringe.

8. The apparatus for extracting nucleic acid according to any of claims 1 to 7, wherein a third valve (108) is provided between the second container (102) and the gas storage means (103).

9. The apparatus for nucleic acid isolation according to any one of claims 1 to 7, wherein the first container (101) is tubular, and is provided at one end with a first valve (106) for connection to the driving means (111) and at the other end with a second valve (107) for connection to the second container (102).

10. The apparatus for extracting nucleic acid according to claim 9, wherein the first container (101) has the following feature (3-1) or (3-2):

(3-1) the first container (101) contains a lysis solution for extracting nucleic acid, a washing solution and an eluent;

alternatively, the first and second electrodes may be,

(3-2) the first container (101) contains a washing solution and an eluent for extracting nucleic acid.

11. The apparatus for extracting nucleic acid according to claim 10, wherein the lysate for extracting nucleic acid is a cell lysate.

12. The apparatus for extracting nucleic acid according to claim 10, wherein the washing solution is a nucleic acid washing solution.

13. The apparatus for extracting nucleic acid according to claim 10, wherein the eluent is a nucleic acid eluent.

14. The apparatus for extracting nucleic acid according to claim 10, wherein the washing solution and the eluting solution are separated from each other.

15. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the washing solution and the eluting solution are arranged in the first container (101) from the proximal end to the distal end at a distance from the second valve (107).

16. The apparatus for extracting nucleic acid according to any one of claims 10 to 14, wherein the washing solution and the eluting solution are separated by a first spacer.

17. The apparatus for extracting nucleic acid according to claim 16, wherein the first container (101) contains a washing solution, a first spacer and an eluent, and they are arranged in the first container (101) in order from the proximal end to the distal end at a distance from the second valve (107).

18. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the washing solution comprises one or more fractions, and the fractions are separated from each other by an additional spacer.

19. The apparatus for extracting nucleic acid according to claim 18, wherein the respective portions have the same or different compositions.

20. The apparatus for extracting nucleic acid according to claim 17, wherein a second spacer is provided between the eluent and the first valve (106).

21. The apparatus for extracting nucleic acid according to claim 20, wherein the first spacer and the second spacer are the same or different.

22. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the second container (102) contains a lysis solution for extracting nucleic acid.

23. The apparatus for extracting nucleic acid according to claim 22, wherein the lysate used for extracting nucleic acid is a cell lysate.

24. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the lysis solution, the washing solution and the elution solution are separated from each other.

25. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the lysis solution, the washing solution and the elution solution are arranged in the first container (101) from the proximal end to the distal end at a distance from the second valve (107).

26. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the lysis solution and the washing solution are separated by a first spacer.

27. The apparatus for extracting nucleic acid according to any one of claims 10 to 14, wherein the washing solution and the eluting solution are separated by a second spacer.

28. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the first container (101) contains a lysis solution, a first spacer, a washing solution, a second spacer and an elution solution, and they are arranged in the first container (101) in order from the proximal end to the distal end at a distance from the second valve (107).

29. The apparatus for extracting nucleic acid according to claim 28, wherein the first spacer and the second spacer are the same or different.

30. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the washing solution comprises one or more fractions, and the fractions are separated from each other by an additional spacer.

31. The apparatus for extracting nucleic acid according to claim 30, wherein the respective portions have the same or different compositions.

32. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein a third spacer is provided between the eluent and the first valve (106).

33. The apparatus for extracting nucleic acid according to any of claims 10 to 14, wherein the first container (101) has a first chamber, a second chamber and a third chamber which are separated from each other; wherein the first chamber is used for containing or containing lysis solution for extracting nucleic acid; the second chamber is used for containing or containing a washing solution for extracting nucleic acid; the third chamber is used for containing or containing an eluent for extracting nucleic acid; and, the second valve (107) is capable of controlling the type of reagent flowing into the second container (102); alternatively, the first and second electrodes may be,

the first container (101) has a first chamber and a second chamber separated from each other; wherein the first chamber is used for containing or containing a washing solution for extracting nucleic acid; the second chamber is used for containing or containing an eluent for extracting nucleic acid; and the second valve (107) is capable of controlling the type of reagent flowing into the second container (102).

34. The apparatus for extracting nucleic acid according to claim 33, wherein the second container (102) contains a lysis solution for extracting nucleic acid.

35. The apparatus for extracting nucleic acid according to claim 34, wherein the lysate is a cell lysate.

36. The device for extracting nucleic acids according to any one of claims 10 to 14, characterized in that it has one or more technical features selected from the group consisting of:

(1) a first valve (106) for controlling fluid communication of the first container (101) with the drive means (111);

(2) the sample outlet (105) is connected with a waste liquid pool (112), or a collecting pool (113), or the waste liquid pool (112) and the collecting pool (113) through a fifth valve (110).

37. The apparatus for extracting nucleic acid according to claim 36, wherein the apparatus further has a driving means (111) in fluid communication with the first container (101) through the first valve (106).

38. The apparatus for extracting nucleic acid according to claim 37, wherein the driving means (111) is detachable.

39. The apparatus for extracting nucleic acid according to claim 37, wherein the driving means (111) is a driving pump.

40. The apparatus for extracting nucleic acid according to claim 36, wherein the fifth valve (110) is capable of controlling a flow direction of a fluid flowing through the fifth valve (110).

41. The apparatus for extracting nucleic acid according to claim 36, wherein the collection well (113) contains a reagent for detecting nucleic acid.

42. The apparatus for extracting nucleic acid according to claim 36, wherein the waste liquid tank (112) and/or the collection tank (113) is detachable.