CN212864720U

CN212864720U - Device for extracting nucleic acid

Info

Publication number: CN212864720U
Application number: CN202020357372.0U
Authority: CN
Inventors: 占伟; 胡陈超; 郭玉淅; 杨毅; 曹甜甜
Original assignee: XIAMEN ZEESAN BIOTECH CO Ltd
Current assignee: XIAMEN ZEESAN BIOTECH CO Ltd
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2021-04-02
Anticipated expiration: 2030-03-20

Abstract

The utility model relates to a device for extracting nucleic acid, it contains: the kit comprises a first container, a second container and a third container, wherein the first container comprises a first sealing reagent layer, a lysis liquid layer and magnetic beads, the first sealing reagent layer is positioned above the lysis liquid layer, and the magnetic beads are positioned in the lysis liquid layer; the second container comprises a second sealing reagent layer and a nucleic acid washing solution layer, and the second sealing reagent layer is positioned above the nucleic acid washing solution layer; and the third container comprises a third sealing reagent layer and a nucleic acid eluent layer, and the third sealing reagent layer is positioned above the nucleic acid eluent layer.

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 obviously reduce the possibility of cross contamination in the process of extracting nucleic acid.

Background

At present, the extraction of nucleic acid becomes the basis for scientific research of all subjects in the field of biomedicine, even agriculture, forestry, animal husbandry, fishery and the like. Whether high-quality nucleic acid can be extracted or not is the key in molecular biology tests, and the sensitivity of the extraction method and the accuracy of the extraction result are directly related to subsequent tests. Therefore, the nucleic acid extraction method is continuously improved and perfected. At present, the methods widely used for nucleic acid extraction include the classical phenol chloroform extraction method, the newly developed silica gel membrane adsorption method, the magnetic bead separation method and the like.

In view of the need for detection over a wide range or simultaneous detection of multiple biological samples, automated devices are currently used in many cases for nucleic acid extraction. However, it has been found that cross-contamination between samples remains a significant problem during nucleic acid extraction of multiple samples using automated devices.

Therefore, there is a need for further improvements in methods and devices for extracting nucleic acids to avoid or reduce cross-contamination between samples.

Disclosure of Invention

In order to solve the above problems, the inventors of the present application have repeatedly searched for and developed a kit and a device for extracting nucleic acid, which can significantly reduce the possibility of cross-contamination between samples during the extraction of nucleic acid, and reduce or even eliminate the detection result of false positive. Based on the method, the application also provides a method for extracting the nucleic acid, and the extraction process is simple and convenient and is more suitable for automatic operation.

Accordingly, in a first aspect of the present application, there is provided a kit for extracting nucleic acid, wherein the kit comprises a sealing reagent and a reagent for extracting nucleic acid, the sealing reagent has a density in a liquid state that is lower than that of the reagent for extracting nucleic acid, and the sealing reagent is immiscible with the reagent for extracting nucleic acid in a liquid state.

In certain embodiments, the sealing reagent is a liquid, e.g., a liquid immiscible with an aqueous solution, e.g., an organic immiscible with water, e.g., a water immiscible, non-volatile organic.

In certain embodiments, the sealing reagent is a solid and is capable of being converted to a liquid under predetermined conditions, and the liquid has a density that is less than the density of the reagent for extracting nucleic acids, and the liquid is immiscible with the reagent for extracting nucleic acids (e.g., it is immiscible with an aqueous solution and is not volatile). In certain embodiments, the predetermined condition is selected from heating, light irradiation and/or chemical treatment. For example, in certain embodiments, the sealing reagent is a solid and is capable of being converted to a liquid under conditions of heating, and the liquid has a density that is less than the density of the reagent for extracting nucleic acids, and the liquid is immiscible with the reagent for extracting nucleic acids (e.g., it is immiscible with aqueous solutions and not volatile).

For example, in certain embodiments, the sealing reagent is a solid below temperature T1 (including T1) and a liquid above temperature T2 (including T2), with T2 > T1. Thus, by heating the sealing agent to a temperature of T2 or more, the sealing agent in a solid state can be liquefied to form a sealing agent in a liquid state. By cooling the sealing agent to a temperature of T1 or less, the liquid sealing agent can be solidified to form a solid sealing agent. In certain embodiments, T1 is 20 to 40 ℃, e.g., 30 to 40 ℃. In certain embodiments, T2 is 45 to 95 ℃, e.g., 60 to 90 ℃, e.g., 70 to 80 ℃.

In certain embodiments, the sealing agent is selected from: mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, or any combination thereof.

As used herein, the term "immiscible" means that two or more liquids do not dissolve in each other when placed in the same container, and spontaneously create a boundary interface, forming a layer when they come into contact.

As used herein, the term "layering" refers to the spontaneous formation of a vertically distributed structure due to the density difference when two or more immiscible liquids are placed in the same container, and the liquid with high density sinks while the liquid with low density floats upwards to form a layer.

As used herein, the sealing reagent of the present invention is immiscible with a reagent for extracting nucleic acid in a liquid state, and has a density lower than that of the reagent for extracting nucleic acid. Therefore, when the sealing reagent of the present invention is in the same container as the reagent for extracting nucleic acid in a liquid state, the two will form a layered structure, and the sealing reagent of the present invention will be located at the upper layer, isolating (i.e., sealing) the reagent for extracting nucleic acid (and other substances, such as nucleic acid, which may be contained therein) located at the lower layer, preventing both the escape of the lower layer reagent (and other substances, such as nucleic acid, which may be contained therein) to the external environment and the entry and contamination of the lower layer by substances in the external environment. In the present application, it is preferred that the nucleic acid molecule is not soluble in the sealing reagent of the present invention. Therefore, the sealing agent of the present invention can exert a better isolation effect. Further, it is to be understood that any substance capable of achieving this function may be used as the sealing agent of the present application, and therefore, the sealing agent of the present application is not limited to the above-listed substances.

In certain embodiments, the reagents for extracting nucleic acids comprise one or more selected from the group consisting of: a lysis solution (e.g., a cell lysis solution), a nucleic acid wash solution, and a nucleic acid eluent.

In certain preferred embodiments, the reagent for extracting nucleic acid comprises a cell lysate, a nucleic acid washing solution, and a nucleic acid eluent, which are placed separately from each other.

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.

In certain embodiments, the kit further comprises a magnet (e.g., a magnetic bead) capable of adsorbing the nucleic acid. In certain embodiments, the kit further comprises a means capable of attracting a magnet (e.g., a magnetic bar, a magnetic rack, etc.).

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.

In certain embodiments, the nucleic acid comprises DNA or RNA.

As used herein, the term "nucleic acid" refers to 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.

In certain embodiments, the kit comprises:

a first container containing a lysate (e.g., a cell lysate);

a second container containing a nucleic acid washing solution;

a third container containing a nucleic acid eluent;

a fourth container containing a sealing reagent.

In certain embodiments, the magnet is placed separately, or, alternatively, in the first container. In certain embodiments, the magnet is disposed at the bottom of the first container.

In certain embodiments, the first, second, third, and/or fourth containers are each independently a desired shape, such as a tube.

In certain embodiments, the first, second, third, and fourth containers are each independently disposed, or are disposed in parallel. For example, the first, second, third and fourth containers are juxtaposed in sequence.

In some embodiments, the first container, the second container and the third container are arranged side by side in sequence, and the relative position relationship among the three containers is fixed. Alternatively, the two containers may be connected by a connecting portion to maintain the positional relationship with each other.

In certain embodiments, the first, second, third, and/or fourth containers are each independently closed. For example, the first, second, third and/or fourth containers may each independently be sealed by a sealing membrane or cap.

In certain embodiments, the kit comprises:

a first container containing a first sealing reagent and a lysis solution (e.g., a cell lysate);

a second container containing a second sealing reagent and a nucleic acid washing solution;

a third container containing a third sealing reagent and a nucleic acid eluent.

In certain embodiments, the first, second, and/or third containers are each independently a desired shape, such as a tube.

In certain embodiments, the first, second and third containers are each independently disposed, or, disposed side-by-side. For example, the first, second and third containers are juxtaposed in sequence.

In certain embodiments, the first, second, and third containers are each independently closed. For example, the first, second and/or third containers may each independently be sealed by a sealing membrane or cap.

In certain embodiments, the first, second, and third sealing reagents are each independently the same or different.

In certain embodiments, the ratio of the volume of nucleic acid eluent to the volume of third sealing reagent is less than 1:1. In certain preferred embodiments, the ratio of the volume of nucleic acid eluting solution to the volume of third sealing reagent is less than 1:1.5, e.g., a volume ratio of 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:5, 1: 10.

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.

Magnetic beads commonly used in nucleic acid extraction in the art are silica membrane-coated magnetic beads, as well as amino magnetic beads on which amino group modification is performed, hydroxyl magnetic beads on which hydroxyl group modification is performed, aldehyde magnetic beads on which aldehyde group modification is performed, and cellulose-coated magnetic beads. The magnetic beads of the present invention include, but are not limited to, the above-mentioned magnetic beads.

In a second aspect of the present application, there is provided a method of extracting nucleic acid, the method comprising, using a kit as described above.

In certain embodiments, the method comprises:

(1) providing a sample comprising nucleic acids;

(2) transferring the sample to a first container, and contacting the sample with a lysis solution in the first container, wherein the lysis solution contains magnetic beads;

(3) taking out the magnetic beads in the previous step, transferring the magnetic beads to a second container, and contacting the magnetic beads with a nucleic acid washing solution in the second container;

(4) and (3) taking out the magnetic beads in the previous step, transferring the magnetic beads to a third container, and contacting the magnetic beads with the nucleic acid eluent in the third container, thereby extracting the nucleic acid into the nucleic acid eluent.

In certain embodiments, in step (2), the first sealing reagent remains positioned above the lysate and isolates (i.e., seals) the lysate (and other substances, e.g., nucleic acids, that may be contained therein).

In certain embodiments, in step (3), the second sealing reagent remains positioned above the nucleic acid wash solution and isolates (i.e., seals) the nucleic acid wash solution (and other substances, e.g., nucleic acids, that may be contained therein).

In certain embodiments, in step (4), the third sealing reagent remains positioned above the nucleic acid eluate and isolates (i.e., seals) the nucleic acid eluate (and other substances, e.g., nucleic acids, that may be contained therein).

In certain embodiments, step (4) is performed under shaking conditions. In certain embodiments, step (4) is performed under shaking conditions and the third sealing reagent forms a water-in-oil structure with the nucleic acid eluent, wherein the third sealing reagent surrounds the nucleic acid eluent.

In certain embodiments, the ratio of the volume of nucleic acid eluent to the volume of third sealing reagent is less than 1:1. In certain preferred embodiments, the ratio of the volume of nucleic acid eluting solution to the volume of third sealing reagent is less than 1:1.5, e.g., a volume ratio of 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:5, 1: 10. In such embodiments, since the volume of the nucleic acid eluent is smaller (or much smaller) than that of the third sealing reagent, and they are immiscible, the nucleic acid eluent is suspended in the third sealing reagent in the form of droplets under the shaking condition, so as to form a state that the third sealing reagent completely encloses the nucleic acid eluent.

In a third aspect of the present application, there is provided a method of extracting nucleic acid, the method comprising:

(1) providing a sample comprising nucleic acids;

(2) contacting the sample with a lysis solution (e.g., a cell lysis solution), a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids, in the presence of a first sealing reagent, wherein the first sealing reagent has a density less than and is immiscible with the lysis solution;

(3) removing the magnet (e.g., magnetic bead) in the previous step, and contacting the magnet (e.g., magnetic bead) with a nucleic acid washing solution in the presence of a second sealing reagent, wherein the second sealing reagent has a density less than and is immiscible with the nucleic acid washing solution;

(4) removing the magnet (e.g., magnetic bead) from the previous step, and contacting the magnet (e.g., magnetic bead) with a nucleic acid eluent in the presence of a third sealing reagent, wherein the third sealing reagent has a density less than and is immiscible with the nucleic acid eluent; thereby, the nucleic acid is extracted into the nucleic acid eluate.

In step (1) of the methods of the present application, the nucleic acid-containing sample can be obtained from any source, including, but not limited to, animals (e.g., cells, tissues, organs, and bodily fluids), plants (e.g., cells, organelles, and tissues), fungi, bacteria, viruses, protists, and environmental samples (e.g., drinking water, sewage, seawater, etc.).

In the method of the present application, any one or any several of the steps (2) to (4) may be operated manually or by an automated device. In certain embodiments of the present application, steps (2) to (4) are performed using an automated apparatus (e.g., a nucleic acid extractor).

In certain embodiments, in step (2) of the methods of the present application, the sample may be contacted with a lysis solution and a magnet using a pipette or any tool capable of holding a nucleic acid sample below the surface of the sealing reagent.

In certain embodiments, in steps (3) and (4) of the methods of the present application, the magnet (e.g., magnetic beads) is removed from the lysate and contacted with the wash solution by a means capable of attracting the magnet (e.g., magnetic bar, magnetic rack); and, the magnet (e.g., magnetic beads) is removed from the wash solution and contacted with the eluent.

In certain embodiments, step (4) is performed under shaking conditions. In certain embodiments, step (4) is performed under shaking conditions and the third sealing reagent forms a water-in-oil structure with the nucleic acid eluent, wherein the third sealing reagent surrounds the nucleic acid eluent. In certain embodiments, in step (4) of the methods of the present application, the nucleic acid eluate is suspended in the form of droplets in the third sealing reagent. In certain preferred embodiments, the nucleic acid eluate is suspended in the third sealing reagent in the form of droplets. In certain preferred embodiments, the nucleic acid eluate is suspended in the third sealing reagent in the form of droplets.

It will be readily appreciated that the above detailed description of the sealing reagent, lysis solution, wash solution, elution solution, magnet, nucleic acid, etc. in the first aspect is equally applicable to the method described in the third aspect.

Thus, in certain embodiments, the first, second, and third sealing reagents are each independently the same or different.

In certain embodiments, the first, second, and third sealing reagents are each independently liquids immiscible with aqueous solutions, e.g., water immiscible organics, e.g., water immiscible, non-volatile organics.

In certain embodiments, the first, second, and third sealing reagents are each independently selected from: mineral oil, animal oil, vegetable oil, synthetic oil, paraffin, silicone oil, or any combination thereof.

In certain embodiments, prior to performing step (2), providing a first sealing reagent, a lysis solution (e.g., a cell lysis solution), and a magnet (e.g., a magnetic bead) capable of adsorbing nucleic acids in a first container, wherein the first sealing reagent is layered with the lysis solution and is located on top of the lysis solution; and, the magnet is located at the lysis liquid layer.

In certain embodiments, prior to performing step (3), a second sealing reagent and a nucleic acid wash solution are provided in a second container, wherein the second sealing reagent forms a layer with the nucleic acid wash solution and is located on top of the nucleic acid wash solution. In certain embodiments, step (3) is performed one or more times (e.g., 1, 2, or 3 times).

In certain embodiments, prior to performing step (4), a third sealing reagent and a nucleic acid eluent are provided in a third container, wherein the third sealing reagent is layered with and is located on top of the nucleic acid eluent. In certain embodiments, step (4) is performed one or more times (e.g., 1, 2, or 3 times).

In certain embodiments, the sample comprising nucleic acids is a sample comprising cells.

In certain embodiments, the nucleic acid comprises DNA or RNA.

In certain embodiments, step (2) through step (4) are performed using an automated apparatus (e.g., a nucleic acid extractor).

In a fourth aspect of the present application, there is provided the use of a kit as described above for extracting nucleic acids.

In a fifth aspect of the present application, there is provided an apparatus for extracting nucleic acid, comprising: a first container 101, a second container 102 and a third container 103, wherein,

first container 101 comprises first sealing reagent layer 104, lysis liquid layer 105 and magnetic beads 106, and first sealing reagent layer 104 is located above lysis liquid layer 105 and magnetic beads 106 are located in lysis liquid layer 105;

the second container 102 contains a second sealing reagent layer 107 and a nucleic acid washing solution layer 108, and the second sealing reagent layer 107 is located above the nucleic acid washing solution layer 108; and

the third container 103 includes a third sealing reagent layer 109 and a nucleic acid eluent layer 110, and the third sealing reagent layer 109 is positioned above the nucleic acid eluent layer 110.

It will be readily appreciated that the above detailed description of the sealing reagents, lysis solutions, wash solutions, eluents, magnets, nucleic acids, etc. of the first aspect is equally applicable to the device described in the fifth aspect.

In certain embodiments, the first container 101, the second container 102, and/or the third container 103 are tubular.

In certain embodiments, the first container 101, the second container 102, and the third container 103 are juxtaposed, e.g., in sequence.

In certain embodiments, the first container 101, the second container 102, and the third container 103 are each independently closed or unsealed.

In certain embodiments, the first container 101, the second container 102, and the third container 103 are closed by a sealing film 111.

Advantageous effects of the invention

The nucleic acid extraction device, the kit and the method provided by the application can obviously reduce the possibility of cross contamination among samples in the nucleic acid extraction process, and reduce or even eliminate false positive detection results. In addition, the nucleic acid extraction device and the method are easy to operate, high in safety, suitable for automatic implementation and capable of effectively preventing environmental pollution and cross contamination among samples.

Drawings

FIG. 1 shows a nucleic acid extraction apparatus (reagent container) used in an experimental group, which comprises a first container/first tube 101, a second container/second tube 102 and a third container/third tube 103 placed side by side, and a sealing film 111 which is positioned above the first container/first tube 101, the second container/second tube 102 and the third container/third tube 103 and seals the mouths thereof; wherein first container/first tube 101 comprises first sealing reagent layer 104, lysis liquid layer 105 and magnetic beads 106, and wherein first sealing reagent layer 104 is located above lysis liquid layer 105 and magnetic beads 106 are located in lysis liquid layer 105; the second container/second tube 102 contains a second sealing reagent layer 107 and a nucleic acid washing solution layer 108, and the second sealing reagent layer 107 is located above the nucleic acid washing solution layer 108; the third container/third tube 103 includes a third sealing reagent layer 109 and a nucleic acid eluent layer 110, and the third sealing reagent layer 109 is positioned above the nucleic acid eluent layer 110.

FIG. 2 shows a nucleic acid extraction apparatus (reagent container) used in the control group, which comprises a first tube 201, a second tube 202 and a third tube 203 placed side by side, and a sealing film 208 which is positioned above the first tube 201, the second tube 202 and the third tube 203 and seals the mouths thereof; wherein the first tube 201 comprises a lysate layer 204 and magnetic beads 205, and the magnetic beads 205 are located in the lysate layer 204; the second tube 202 comprises a nucleic acid wash solution layer 206; the third tube 203 contains a nucleic acid eluent layer 207.

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: nucleic acid extraction process

1.1 Pre-filling of reagents

As shown in fig. 1, the following reagents were pre-filled in reagent containers consisting of 3 tubes of 3 ml: the first tube 101 contains 1ml of lysis solution 105 (4M guanidine hydrochloride, 50mM Tris-HCl, 10mM EDTA, 15% Triton X100, pH 6.5) and silica gel membrane magnetic beads 106 (containing magnetite and silica) in the lower layer, and 0.5ml of first sealing reagent 104 in the upper layer, the first sealing reagent 104 being mineral oil (available from sigma aldrich trade ltd, cat # M8410); the second tube 102 contains 1ml of a wash solution 108 (100 mM NaCl, 50mM Tris-HCl (pH 7.8), 75% absolute ethanol) in the lower layer, and 0.5ml of a second sealing reagent 107 (mineral oil, available from sigma aldrich trade, ltd., product number M8410) in the upper layer; the third tube 103 contains 0.1ml of eluent 110 (formulation 10mM Tris-HCl, 1mM EDTA, pH 8.5) in the lower layer and 0.3ml of a third sealing reagent 109 in the upper layer, mineral oil (available from sigma aldrich trade limited, cat # M8410).

The top of the reagent container is sealed 111 with a sealing film.

1.2 sample addition

The sealing film 111 of the reagent container is torn off, and then 0.5ml of plasmid DNA sample is pipetted into the lower layer reagent of the first tube 101 (the tip of the pipetter needs to penetrate the upper layer of the first sealing reagent 104).

1.3 extracting on machine

Nucleic acid extraction was performed using a nucleic acid extractor (Shiyasu Biotechnology Ltd., Lab-Aid 824s nucleic acid extractor, cat # 501007) according to the manufacturer's instructions. Briefly, the reagent vessel was placed in the nucleic acid extractor, the magnetic sleeve was inserted according to the instructions, the instrument operating software was opened, the "NA-DNA" program was called, and the "run" button was clicked to execute the program. The extraction procedure is set up as follows: sample cracking, magnetic bead and nucleic acid combination, washing a compound of the magnetic bead and the nucleic acid, and eluting the nucleic acid. The instrument automatically completes the extraction of nucleic acid from the sample.

After the automatic operation of the instrument is finished, the extracted nucleic acid is sucked out by using a liquid transfer device for later use. When the nucleic acid is aspirated, it is necessary to take care that the elution solution in which the nucleic acid is dissolved is located in a lower layer of the sealing reagent or suspended in the form of droplets in the sealing reagent.

Example 2: comparison experiment of anti-pollution effect in nucleic acid extraction process

2.1 comparative Experimental setup

As shown in tables 1 and 2, the experiment was divided into a control group and an experimental group, each group was provided with 7 subgroups (i.e., 4 full-negative groups +3 positive-negative groups), each subgroup having 24 samples; wherein all samples used in the total negative group are negative samples; also, in the yin-yang group, negative and positive samples were crossed (as shown in table 3). In this experiment, the positive sample was plasmid DNA and the negative sample was sterilized purified water.

The procedure for extracting nucleic acid from the experimental group was as described in example 1 (FIG. 1), and the sealing reagent was added to the upper layer of each test tube, whereas the sealing reagent was not added to the control group, which was identical to the experimental group (FIG. 2).

TABLE 1 setting of control samples

TABLE 2 set-up of experimental group samples

Experiment number	Group of	Sample(s)	Whether to add sealing reagents
				1	Whole yin group	Full negative (24-)	Is that
2	Yin and yang group	Yin-yang cross (12+/12-)	Is that
				3	Whole yin group	Full negative (24-)	Is that
4	Yin and yang group	Yin-yang cross (12+/12-)	Is that
				5	Whole yin group	Full negative (24-)	Is that
6	Yin and yang group	Yin-yang cross (12+/12-)	Is that
				7	All yinGroup of	Full negative (24-)	Is that

TABLE 3 setting of Yin Yang group samples

2.2 nucleic acid detection

The nucleic acid samples of the experimental group and the control group were each detected in a real-time fluorescence PCR apparatus (Shanghai Hongshi medical science Co., Ltd., model SLAN-96S) according to the instructions of the real-time fluorescence PCR detection kit (Xiamen-Shi Biotechnology Co., Ltd., product No. 801010).

2.3 results of the experiment

The results of the measurements are shown in tables 4 and 5. As can be seen from Table 4, cross contamination of different degrees occurred in all of the 3 yin-yang groups set in the control group, which is caused by inaccurate detection results due to the fact that part of the negative samples are contaminated by the nucleic acid of the positive samples without adding the sealing reagent. The test groups to which the sealing reagent was added were accurately detected for both negative and positive samples (Table 5), which confirmed that the sealing solution was able to prevent cross-contamination between nucleic acid samples.

TABLE 4 test results of control samples

TABLE 5 test results of the samples of the experimental groups

It is noted that in practical applications, with the large increase of the extraction amount of the nucleic acid sample, cross contamination between the whole batch of samples may also occur during the extraction process without adding a sealing reagent.

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. A device for extracting nucleic acids, comprising: a first container (101), a second container (102) and a third container (103), wherein,

the first container (101) comprises a first sealing reagent layer (104), a lysis liquid layer (105) and magnetic beads (106), and the first sealing reagent layer (104) is located above the lysis liquid layer (105) and the magnetic beads (106) are located in the lysis liquid layer (105);

the second container (102) comprises a second sealing reagent layer (107) and a nucleic acid washing solution layer (108), and the second sealing reagent layer (107) is positioned above the nucleic acid washing solution layer (108); and

the third container (103) contains a third sealing reagent layer (109) and a nucleic acid eluent layer (110), and the third sealing reagent layer (109) is located above the nucleic acid eluent layer (110).

2. The device according to claim 1, characterized in that the first container (101), the second container (102) and/or the third container (103) are tubular.

3. The device according to claim 1, characterized in that said first container (101), second container (102) and third container (103) are juxtaposed.

4. The apparatus of claim 3, wherein the juxtaposition is sequential.

5. The device according to any one of claims 1 to 4, wherein the first container (101), the second container (102) and the third container (103) are each independently closed or not closed.

6. Device according to any of claims 1 to 4, wherein the first container (101), the second container (102) and the third container (103) are closed by a sealing film (111).