CN113930336A - Nucleic acid extraction kit by magnetic bead method and extraction method thereof - Google Patents
Nucleic acid extraction kit by magnetic bead method and extraction method thereof Download PDFInfo
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- CN113930336A CN113930336A CN202111275544.5A CN202111275544A CN113930336A CN 113930336 A CN113930336 A CN 113930336A CN 202111275544 A CN202111275544 A CN 202111275544A CN 113930336 A CN113930336 A CN 113930336A
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The invention discloses a nucleic acid extraction kit by a paramagnetic particle method and an extraction method thereof, and the nucleic acid extraction kit comprises a box body (1), wherein a sample adding cavity (2) and a mixing cavity (3) are arranged on the box body (1), and a connecting liquid path (4) is arranged between the sample adding cavity (2) and the mixing cavity (3); the mixing cavity (3) is provided with an air hole (31), and the sample adding cavity (2) is provided with a sample adding hole (21). When the kit is used for extracting nucleic acid of a sample, only a plurality of steps need to be completed in one kit, external pollution is not easy to introduce, and the extraction efficiency can be greatly improved.
Description
Technical Field
The invention relates to a nucleic acid extraction kit and an extraction method by a paramagnetic particle method, and belongs to the technical field of medical detection.
Background
With the popularization of technical means such as gene detection and the like, the limitation of the traditional nucleic acid extraction method is very obvious today when high throughput and automation are pursued. At present, the magnetic bead method for extracting nucleic acid can realize automatic extraction and large-batch operation, is simple to operate and short in time, so that the magnetic bead method for extracting nucleic acid is more and more frequently used in the field of medical detection.
The general steps for extracting nucleic acids by the magnetic bead method are lysis or conversion, washing and elution. At present, a plurality of steps for extracting nucleic acid by a magnetic bead method are generally carried out in different test tubes or kits, a plurality of test tubes are required to be prepared, samples are required to be continuously transferred in the extraction process, external pollution is easily introduced, the operation is more complicated on the other hand, and the extraction efficiency is lower.
Disclosure of Invention
The invention aims to provide a nucleic acid extraction kit and an extraction method by a magnetic bead method. When the kit is used for extracting nucleic acid of a sample, only a plurality of steps need to be completed in one kit, external pollution is not easy to introduce, and the extraction efficiency can be greatly improved.
The technical scheme of the invention is as follows: a nucleic acid extraction kit by a magnetic bead method is characterized in that: the device comprises a box body, wherein a sample adding cavity and a mixing cavity are arranged on the box body, and a connecting liquid path is arranged between the sample adding cavity and the mixing cavity; the mixing chamber is provided with an air hole, and the sample adding chamber is provided with a sample adding hole.
In the kit for extracting nucleic acid by using the paramagnetic particle method, the connecting liquid path is respectively connected with the lower ends of the sample adding cavity and the mixing cavity.
In the kit for extracting nucleic acid by the paramagnetic particle method, the lower ends of the sample adding cavity and the mixing cavity are provided with inverted triangular pyramids. Wherein the inverted triangular cone area of the lower end of the mixing cavity is used as a magnetic bead adsorption area for adsorbing magnetic beads in the funnel-shaped area when the magnetic beads are close to the magnet.
In the kit for extracting nucleic acid by using the magnetic bead method, the bottoms of the sample adding cavity and the mixing cavity are obliquely guided and connected with the liquid path.
The extraction method of the nucleic acid extraction kit by using the magnetic bead method is characterized by comprising the following steps of:
adding a binding solution, a magnetic bead solution and a sample solution subjected to cracking or conversion into a sample adding cavity from a sample adding hole;
the air hole is connected with an air pump, the solution added from the sample adding cavity flows back and forth between the mixing cavity and the sample adding cavity by controlling the positive pressure and the negative pressure of the air pump in a reciprocating manner, and all the solution is sucked into the mixing cavity after the solution is fully and uniformly mixed;
and thirdly, the magnet absorbs the magnetic beads in the solution in the magnetic bead absorption area of the mixing cavity through magnetic force, the mixture is kept still for a period of time, and after the solution in the mixing cavity becomes clear, the air pump pumps air to extrude the supernatant to the sample adding cavity and move the supernatant away by using a pipettor.
In the foregoing extraction method, further, after the step (c), a cleaning step is further included:
fourthly, adding quantitative cleaning fluid into the sample adding cavity, electromagnetically releasing magnetic beads, and repeating the second step and the third step.
In the above extraction method, preferably, the step (iv) is repeated a plurality of times using different or the same washing solutions.
In the above extraction method, the step iv further includes an elution step:
fifthly, adding eluent into the sample adding cavity, releasing the magnetic beads by the magnet, and repeating the step two;
and sixthly, the magnet absorbs the magnetic beads, the mixture is kept stand for a period of time, after the solution in the mixing cavity becomes clear, the air pump pumps air, the supernatant is extruded to the sample adding cavity, and the solution is extracted into a specified test tube by a pipettor.
Compared with the prior art, the kit provided by the invention has the advantages that the two chambers communicated by the connecting liquid path are respectively used as the sample adding chamber and the mixing chamber, the solution added into the sample adding chamber can freely move between the two chambers by utilizing the pneumatic principle, the liquid transfer can be realized, and the liquid can be uniformly mixed by repeatedly transferring the liquid back and forth. Therefore, the kit can sequentially carry out the steps of magnetic bead combination, one-time or multiple-time cleaning, elution and the like on the sample, different reagents are sequentially added into the sample adding cavity, sample nucleic acid does not need to be transferred, the whole extraction process can be finished in one reagent kit, the pollution of an external machine is not easy to introduce, the operation is simple, and only the action of an air pump and the addition/derivation of the reagents need to be controlled.
On the other hand, the invention also provides a using method for the kit, the method is extremely simple to operate, the automation of time extraction by external equipment is easy to utilize, the external equipment only needs to have the functions of air suction, reagent addition/removal and the like, the implementation is easy, and the working efficiency is higher.
Drawings
FIG. 1 is a schematic structural diagram of a kit according to example 1 of the present invention;
FIG. 2 is a schematic structural diagram of a kit according to example 2 of the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Example 1: a nucleic acid extraction kit by a magnetic bead method is shown in figure 1: the device comprises a box body 1, wherein a sample adding cavity 2 and a mixing cavity 3 are arranged on the box body 1, and a connecting liquid path 4 is arranged between the sample adding cavity 2 and the mixing cavity 3; the mixing cavity 3 is provided with an air hole 31, and the sample adding cavity 2 is provided with a sample adding hole 21. The connecting liquid path 4 is respectively connected with the lower ends of the sample adding cavity 2 and the mixing cavity 3. The lower ends of the sample adding cavity 2 and the mixing cavity 3 are provided with inverted triangular pyramids. The bottom parts of the sample adding cavity 2 and the mixing cavity 3 are inclined, guided and connected with a liquid path 4.
The extraction method of the nucleic acid extraction kit by using the magnetic bead method comprises the following steps:
adding a binding solution, a magnetic bead solution and a sample solution subjected to cracking or conversion into a sample adding cavity 2 from a sample adding hole 21;
the air hole 31 is connected with an air pump, the solution added from the sample adding cavity 2 flows back and forth between the mixing cavity 3 and the sample adding cavity 2 by controlling the positive pressure and the negative pressure of the air pump in a reciprocating way, and all the solution is sucked into the mixing cavity 3 after the solution is fully and uniformly mixed;
and thirdly, the magnet absorbs the magnetic beads in the solution in the magnetic bead adsorption area 32 of the mixing cavity 3 through magnetic force, the magnet stands for a period of time, and after the solution in the mixing cavity 3 becomes clear, the air pump pumps air to extrude the supernatant to the sample adding cavity 2 and move the supernatant away by using a pipettor.
Fourthly, adding quantitative cleaning fluid into the sample adding cavity, electromagnetically releasing magnetic beads, and repeating the second step and the third step.
And fourthly, repeating the step IV by using different or same cleaning liquids for multiple times according to requirements.
Fifthly, eluent is added into the sample adding cavity 2, the magnetic beads are released by the magnet, and the second step is repeated;
sixthly, the magnet attracts the magnetic beads, standing for a period of time, after the solution in the mixing cavity 3 becomes clear, pumping air by an air pump, extruding the supernatant into the sample adding cavity 2, and extracting the solution into a specified test tube by a pipettor.
Example 2: a nucleic acid extraction kit by a magnetic bead method is shown in figure 2: the difference from example 1 is that there is an addition well 33 above the mixing chamber for adding the magnetic bead solution, and the well must be sealed after addition.
The specific operation steps are as follows:
adding a binding solution and a sample solution subjected to cracking or conversion into a sample adding cavity 2 from a sample adding hole 21; adding a magnetic bead solution into the mixing chamber from the well 33, and sealing the well 33;
the air hole 31 is connected with an air pump, and the solution in the sample adding cavity 2 is pumped into the mixing cavity 3 to be mixed with the magnetic bead solution by controlling the positive pressure and the negative pressure of the air pump;
thirdly, the magnet sucks the magnetic beads in the solution into the magnetic bead adsorption area 32 of the mixing cavity 3 through magnetic force, the mixture is kept still for a period of time, after the solution in the mixing cavity 3 becomes clear, the air pump pumps air, and the supernatant is extruded to the sample adding cavity 2;
fourthly, repeating the steps II and III for a plurality of times, extruding the supernatant into the sample adding cavity 2 and then removing the supernatant by a liquid transfer device.
Fifthly, adding quantitative cleaning liquid into the sample adding cavity, electromagnetically releasing magnetic beads, and repeating the steps II, III and IV.
Repeating the above steps with different or same cleaning solution for several times according to requirement.
Sixthly, eluent is added into the sample adding cavity 2, the magnetic beads are released by the magnet, and the step II and the step III are repeated;
and seventhly, attracting the magnetic beads by a magnet, standing for a period of time, pumping by an air pump after the solution in the mixing cavity 3 becomes clear, extruding the supernatant into the sample adding cavity 2, and extracting the solution into a specified test tube by using a pipettor.
The advantages of this method are: the magnetic beads are always in the mixing cavity, so that the magnetic beads are prevented from remaining in other chambers or liquid paths, and the nucleic acid extraction efficiency is prevented from being reduced.
Claims (8)
1. A magnetic bead method nucleic acid extraction kit is characterized in that: comprises a box body (1), wherein a sample adding cavity (2) and a mixing cavity (3) are arranged on the box body (1), and a connecting liquid path (4) is arranged between the sample adding cavity (2) and the mixing cavity (3); the mixing cavity (3) is provided with an air hole (31), and the sample adding cavity (2) is provided with a sample adding hole (21).
2. The kit for nucleic acid extraction by the magnetic bead method according to claim 1, wherein: and the connecting liquid path (4) is respectively connected with the lower ends of the sample adding cavity (2) and the mixing cavity (3).
3. The kit for nucleic acid extraction by the magnetic bead method according to claim 1, wherein: and the lower ends of the sample adding cavity (2) and the mixing cavity (3) are provided with inverted triangular conical surfaces.
4. The multi-channel nucleic acid secondary amplification kit according to claim 1, characterized in that: the bottom parts of the sample adding cavity (2) and the mixing cavity (3) are inclined, guided and connected with the liquid path (4).
5. The method for extracting nucleic acid using the magnetic bead method nucleic acid extraction kit according to any one of claims 1 to 5, comprising the steps of:
adding a binding solution, a magnetic bead solution and a sample solution subjected to lysis or conversion into a sample adding cavity (2) from a sample adding hole (21);
the air hole (31) is connected with an air pump, the solution added from the sample adding cavity (2) flows back and forth between the mixing cavity (3) and the sample adding cavity (2) by controlling the positive pressure and the negative pressure of the air pump in a reciprocating way, and all the solution is sucked into the mixing cavity (3) after the solution is fully and uniformly mixed;
and thirdly, the magnet sucks the magnetic beads in the solution in the mixing cavity (3) through magnetic force, the magnetic beads are kept still for a period of time, after the solution in the mixing cavity (3) becomes clear, the air pump pumps air, the supernatant is extruded to the sample adding cavity (2), and the sample adding cavity is removed by a liquid transferring device.
6. The extraction method according to claim 5, further comprising, after the step (c), the step of:
fourthly, adding quantitative cleaning fluid into the sample adding cavity, electromagnetically releasing magnetic beads, and repeating the second step and the third step.
7. The extraction method according to claim 6, characterized in that: and fourthly, repeatedly carrying out the step IV by using different or same cleaning solutions for many times.
8. The extraction method according to claim 6, characterized in that the step (iv) is followed by the steps of:
fifthly, adding eluent into the sample adding cavity (2), releasing the magnetic beads by the magnet, and repeating the second step;
and sixthly, the magnetic beads are attracted by a magnet, standing is carried out for a period of time, after the solution in the mixing cavity (3) becomes clear, the air pump pumps air, the supernatant is extruded to the sample adding cavity (2), and the solution is extracted into a designated test tube by a pipettor.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111275544.5A CN113930336A (en) | 2021-10-29 | 2021-10-29 | Nucleic acid extraction kit by magnetic bead method and extraction method thereof |
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| CN202111275544.5A CN113930336A (en) | 2021-10-29 | 2021-10-29 | Nucleic acid extraction kit by magnetic bead method and extraction method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112812959A (en) * | 2019-11-15 | 2021-05-18 | 广州万孚生物技术股份有限公司 | Nucleic acid extraction, amplification and detection integrated device |
| CN112958173A (en) * | 2021-03-04 | 2021-06-15 | 深圳市美好创亿医疗科技股份有限公司 | Microfluidic kit |
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- 2021-10-29 CN CN202111275544.5A patent/CN113930336A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112812959A (en) * | 2019-11-15 | 2021-05-18 | 广州万孚生物技术股份有限公司 | Nucleic acid extraction, amplification and detection integrated device |
| CN112958173A (en) * | 2021-03-04 | 2021-06-15 | 深圳市美好创亿医疗科技股份有限公司 | Microfluidic kit |
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Address after: 225129 building 6, Chuangfu workshop, No. 1, Chuangfu Road, Hanjiang District, Yangzhou City, Jiangsu Province Applicant after: Jiangsu Fuyuan Biotechnology Co.,Ltd. Applicant after: HANGZHOU LIFEREAL BIOTECHNOLOGY Co.,Ltd. Applicant after: Shanghai Fuyuan Biotechnology Co.,Ltd. Applicant after: Hangzhou Suizeng Biotechnology Co.,Ltd. Address before: 225129 building 6, Chuangfu workshop, No. 1, Chuangfu Road, Hanjiang District, Yangzhou City, Jiangsu Province Applicant before: Jiangsu Fuyuan Biotechnology Co.,Ltd. Applicant before: HANGZHOU LIFEREAL BIOTECHNOLOGY Co.,Ltd. Applicant before: SINGLERA GENOMICS (SHANGHAI) Ltd. Applicant before: Hangzhou Suizeng Biotechnology Co.,Ltd. |
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Address after: 225129 building 6, Chuangfu workshop, No. 1, Chuangfu Road, Hanjiang District, Yangzhou City, Jiangsu Province Applicant after: Jiangsu Fuyuan Biotechnology Co.,Ltd. Applicant after: HANGZHOU LIFEREAL BIOTECHNOLOGY Co.,Ltd. Applicant after: Jiangsu Huayuan Biotechnology Co.,Ltd. Applicant after: Hangzhou Suizeng Biotechnology Co.,Ltd. Address before: 225129 building 6, Chuangfu workshop, No. 1, Chuangfu Road, Hanjiang District, Yangzhou City, Jiangsu Province Applicant before: Jiangsu Fuyuan Biotechnology Co.,Ltd. Applicant before: HANGZHOU LIFEREAL BIOTECHNOLOGY Co.,Ltd. Applicant before: Shanghai Fuyuan Biotechnology Co.,Ltd. Applicant before: Hangzhou Suizeng Biotechnology Co.,Ltd. |
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