CN108841818B - Nucleic acid extraction method and device - Google Patents
Nucleic acid extraction method and device Download PDFInfo
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- CN108841818B CN108841818B CN201810203382.6A CN201810203382A CN108841818B CN 108841818 B CN108841818 B CN 108841818B CN 201810203382 A CN201810203382 A CN 201810203382A CN 108841818 B CN108841818 B CN 108841818B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- 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
Abstract
The invention discloses a nucleic acid extraction method and a device, comprising a sample extraction module, a sample storage module and a nucleic acid extraction module, wherein the sample extraction module is used for containing a nucleic acid sample and extracting nucleic acid from the sample; the magnetic bead injection pump is used for introducing a solution mixed with micron magnetic beads into the sample extraction module; the magnetic field generating module is used for generating a high-intensity magnetic field; a sample lysis module for separating nucleic acids from a sample; and the eluent injection pump is used for introducing eluent into the sample extraction module to separate the nucleic acid from the magnetic beads and recovering the magnetic beads to obtain the nucleic acid. The invention provides a method and a device for effectively, quickly and semi-automatically extracting nucleic acid from a large-volume biological sample, which can realize quick extraction of nucleic acid from a sample of more than 10 milliliters, has shorter extraction time compared with the traditional mode, can automatically operate the whole extraction link, and can be combined with other detection devices in the later stage to achieve the aim of full-automatic detection.
Description
Technical Field
The present invention relates to the field of continuous fluid separation. More particularly, it relates to a nucleic acid extraction method and apparatus.
Background
Molecular diagnosis uses nucleic acid as a detection object and is mainly applied to various biological diagnoses. Before molecular diagnosis of a sample, nucleic acid needs to be extracted from a sample to be detected, and the concentration and purity of the nucleic acid directly influence the accuracy of the molecular diagnosis. The traditional extraction of nucleic acid generally comprises density gradient centrifugation, phenol/chloroform, phenol, isoamyl alcohol extraction and other methods, but the methods are limited because the methods are toxic, time-consuming or require large and complicated devices. In recent years, the magnetic bead method has been widely used for extracting nucleic acids from a sample. The general steps of the method are as follows: firstly, cracking nucleic acid of an object to be detected from cells through a cracking solution, then capturing the nucleic acid through the electrostatic action on the surface of magnetic beads, then applying a magnetic field to separate the nucleic acid combined with the magnetic beads from an original sample, then removing the magnetic field, adding eluent, separating the magnetic beads from the nucleic acid, and obtaining the purified nucleic acid through magnetic separation again. Compared with other methods, the method is simpler, more convenient, safer and nontoxic, and the concentration and purity of the extracted nucleic acid are higher.
However, the conventional magnetic bead method for extracting nucleic acid has the following disadvantages: first, the volume of a sample for extracting nucleic acid by the magnetic bead method is limited, and is generally several tens microliters to several hundreds microliters. Secondly, the operation steps of extracting nucleic acid by a manual magnetic bead method are complex, and skilled technicians are required to perform skilled operation; the common automatic nucleic acid extraction device in the market has large volume, is expensive and not easy to carry, and is difficult to meet the requirements of field detection. Finally, the recently developed microfluidic magnetic bead nucleic acid extraction method generally has a small volume of a sample to be detected and a slow extraction speed. In view of the above problems, the present invention provides a method for capturing a large volume of sample by extracting nucleic acid from a continuously flowing sample; a small semi-automated device is provided, which is simple to operate, portable, and fast in capture speed, and can capture DNA in a large number of samples in a short time.
Therefore, it is desirable to provide a method and apparatus for nucleic acid extraction.
Disclosure of Invention
The invention aims to provide a nucleic acid extraction method and a nucleic acid extraction device.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of nucleic acid extraction comprising:
introducing the solution mixed with the micron magnetic beads into a container, and placing the container in a magnetic field to enable the magnetic beads in the solution to be bonded in a chain shape and fill the container;
cracking the sample added with the cracking liquid, and then introducing the sample into the container, so that the nucleic acid in the cracked sample is combined with the magnetic beads in the container and adsorbed in the container, and other impurities flow out of the outlet of the container along with the mixed solution;
introducing a washing solution into the container, and purifying the nucleic acid sample combined with the magnetic beads in the container;
and introducing an eluent into the container to separate the nucleic acid in the nucleic acid sample in the container from the magnetic beads.
Further, the step of introducing the sample added with the lysis solution into the container after the sample is lysed comprises the following steps: and (3) cracking the sample added with the cracking solution for 10 minutes, and then introducing the sample into the container for 3-5 minutes.
Further, a washing solution is mixed with the nucleic acid sample after passing through the container and is maintained for 3 to 5 minutes.
Further, the eluent is mixed with the nucleic acid sample and kept for 3-5 minutes after passing through the container.
The invention also discloses a nucleic acid extraction device, comprising:
the sample extraction module is used for containing a nucleic acid sample and extracting nucleic acid from the sample;
the magnetic bead injection pump is used for introducing a solution mixed with micron magnetic beads into the sample extraction module;
the magnetic field generating module is used for generating a high-intensity magnetic field;
a sample lysis module for separating nucleic acids from a sample;
and the eluent injection pump is used for introducing eluent into the sample extraction module to separate the nucleic acid from the magnetic beads to obtain the nucleic acid.
Furthermore, the sample extraction module is arc-shaped and comprises an inlet, an outlet, a shell and an internal channel, wherein the internal channel is S-shaped and is uniformly distributed in the shell, and two ends of the internal channel are respectively connected with the inlet and the outlet.
Further, the magnetic field generation module is in a hollow arc shape and comprises a large magnet, a small magnet and a base, the large magnet and the small magnet are in arc shapes and are concentrically arranged in the base, the sample extraction module is arranged between the large magnet and the small magnet, and a high-strength magnetic field is generated between the large magnet and the small magnet.
Furthermore, the sample cracking module comprises a cracking channel pipe, a heating pipe, a heat insulation pipe and a temperature probe, wherein the cracking channel pipe is connected with the inlet of the sample extraction module, the heating pipe is sleeved on the outer surface of the cracking channel pipe and heats the cracking channel pipe, the heat insulation pipe is sleeved on the outer surface of the heating pipe and is used for reducing heat dissipation, and the temperature probe is connected with the cracking channel pipe and is used for measuring the temperature of the cracking channel pipe and feeding back the temperature.
Furthermore, the extraction device also comprises a washing solution injection pump, a first multi-way tube, a second multi-way tube and a plurality of multi-way valves, wherein the washing solution injection pump is used for introducing washing solution into the sample extraction module to purify the nucleic acid sample combined with the magnetic beads; one end of the first multi-way tube is communicated with the inlet of the sample extraction module, and the other end of the first multi-way tube is respectively connected with the magnetic bead injection pump, the sample cracking module, the washing solution injection pump and the eluent injection pump; one end of the second multi-way pipe is communicated with the outlet of the sample extraction module, and the other end of the second multi-way pipe is respectively communicated with the extract and the waste liquid recovery unit.
Furthermore, the extraction device also comprises a control module, wherein the control module comprises a control circuit and a microprocessor and is used for controlling the working state of each module in the extraction device and the opening and closing state of the multi-way valve.
The invention has the following beneficial effects:
the technical scheme of the invention provides an effective, rapid and semi-automatic method and device for extracting nucleic acid from a large-volume biological sample, the method can realize rapid extraction of nucleic acid from a sample of more than 10 milliliters, the extraction time is shorter than that of the traditional mode, the whole extraction link can be operated automatically, and the method can be combined with other detection devices in the later period to achieve the purpose of full-automatic detection.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings;
FIG. 1 is a flow chart of a method for extracting nucleic acid according to the present invention;
FIG. 2 is a schematic view of a nucleic acid isolation apparatus according to the present invention;
FIG. 3 is a schematic diagram of a sample extraction module according to the present invention;
FIG. 4 is a schematic structural diagram of a magnetic field generating module according to the present invention;
FIG. 5 is a schematic structural diagram of a sample lysis module provided in the present invention;
FIG. 6 is a schematic view of a part of the nucleic acid isolation apparatus according to the present invention.
Wherein, 1, a sample extraction module; 2. a magnetic field generating module; 3. a control module; 4. a first multi-pass tube; 5. a second multi-pass tube; 6. a sample lysis module; 7. a sample injection pump; 8. a magnetic bead injection pump; 9. a washing liquid injection pump; 10. an eluent injection pump; 11. a nucleic acid collection pump; 12. a waste liquid recovery pump; 13. an inlet; 14. an outlet; 15. a housing; 16. an internal channel; 17. bolt holes, 18, large magnets; 19. a small magnet; 20. a base; 21. a side fixing frame; 22. a channel inlet and outlet conduit; 23. connecting a conduit; 24. a lysis channel tube; 25. a heat insulating pipe; 26. heating a tube; 27. a temperature measuring probe.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
As shown in fig. 1 and fig. 2, the nucleic acid extraction method disclosed by the present invention comprises:
s1, the solution mixed with the micron magnetic beads is introduced into a container, and the container is placed in a magnetic field to enable the magnetic beads in the solution to be combined in a chain shape and fill the container.
The magnetic bead injection pump 8 is turned on, the solution mixed with the micro magnetic beads enters the sample extraction module 1 through the first multi-way tube 4, and under the action of the magnetic field, the solution in the container is combined in a chain shape and fills the whole sample extraction module 1, and the rest solution flows out from the outlet.
S2, cracking the sample added with the cracking liquid, and then introducing the sample into the container, so that the nucleic acid in the cracked sample is combined with the magnetic beads in the container and adsorbed in the container, and other impurities flow out from the outlet of the container along with the mixed solution.
The sample injection pump 7 is started, the sample added with the lysis solution is introduced into the sample lysis module 6, the sample is lysed for 10 minutes, then the sample enters the sample extraction module 1 through the first multi-way tube 4 and is kept for 3-5 minutes (or is injected into the sample extraction module 1 at a lower speed, namely not more than 0.5 mL/min), the nucleic acid after lysis is combined with the surface of the chain-forming magnetic bead of the inner sample extraction module 1 and is adsorbed in the sample extraction module 1, and other impurities in the sample and the like are discharged from an outlet.
S3, introducing a washing solution into the container, and purifying the nucleic acid sample bonded with the magnetic beads in the container.
And starting a washing liquid injection pump 9, introducing the washing liquid into the sample extraction module 1, and keeping the washing liquid for 3-5min (or slowly introducing the washing liquid into the sample extraction module), so that impurities in some samples are continuously separated from the nucleic acid, and the purpose of purification is achieved.
S4, introducing the eluent into the container to separate the nucleic acid in the nucleic acid sample in the container from the magnetic beads.
Starting the eluent injection pump 10, introducing the eluent into the sample extraction module 1 and keeping the eluent for 3min (or injecting the eluent into the channel at a lower speed, namely not more than 0.1 mL/min), separating the magnetic beads from the captured nucleic acids under the action of the eluent, and enabling the separated nucleic acids and the eluent to flow out from the outlet. And after the nucleic acid extraction is finished, taking the sample extraction module 1 out of the magnetic field generation module 2, and introducing a solution to recover the magnetic beads.
The invention also discloses a nucleic acid extraction device, as shown in FIG. 2, comprising: the sample extraction module 1 is used for containing a nucleic acid sample and extracting nucleic acid from the sample;
a magnetic bead injection pump 8 for introducing a solution mixed with micron magnetic beads into the sample extraction module 1;
the magnetic field generating module 2 is used for generating a high-intensity magnetic field;
a sample lysis module 6 for separating nucleic acids from the sample and binding the nucleic acids to magnetic beads;
and an eluent injection pump 10 for introducing eluent into the sample extraction module 1 to separate nucleic acid from magnetic beads to obtain nucleic acid.
Specifically, as shown in fig. 3, the sample extraction module 1 is in the shape of an arc, and includes an inlet 13, an outlet 14, a housing 15, and an internal passage 16, where the internal passage 16 is uniformly arranged in the housing 15 in an S-shape, and two ends of the internal passage are connected to the inlet 13 and the outlet 14, respectively. The housing is circular in shape horizontally, the housing is made of a biochemical inert material, the inner channel 16 is made of an anti-corrosion material, and liquid can flow inside the inner channel.
As shown in fig. 4, the magnetic field generating module 2 is a hollow circular arc, and includes a large magnet 18, a small magnet 19 and a base 20, the large magnet 18 and the small magnet 19 are circular arc-shaped and concentrically arranged in the base 20, the sample extracting module 1 is arranged between the large magnet 18 and the small magnet 19, and is detachably embedded between the large magnet 18 and the small magnet 19, both magnets are high-strength magnets made of neodymium-iron-boron-N52 from N level to S level in the radial direction, and generate a high-gradient and high-strength magnetic field in the radial direction, especially at the position of the sample extracting module 1, and in addition, three bolts are used to fix the side fixing frame on the base 20 in the bolt hole 17. The internal channel 16 of the extraction module 1 is consistent with the arc magnetic field constructed by the magnetic field generation module 2, so that each region in the channel 16 can be ensured to keep the same or similar magnetic field condition, and further, magnetic beads are uniformly linked in the channel 16, and therefore, magnetic beads capable of capturing samples are uniformly distributed in all regions in the channel 16.
As shown in FIG. 5, the sample cracking module 6 comprises a cracking channel tube 24, an insulating tube 25, a heating tube 26 and a temperature probe 27. The cracking channel pipe 24 is connected with one of the connecting conduits of the first multi-way pipe 4, the heating pipe 26 is used for heating the cracking channel pipe 24, the heat insulation pipe 25 can reduce the heat radiation of the cracking channel pipe 24, the temperature probe 27 measures the temperature and feeds the temperature back to the control unit 3, and the control unit 3 controls the heating pipe 26 to work through the measured temperature.
As shown in fig. 6, with reference to fig. 2, the extraction device further includes a washing solution injection pump 9, a side fixing frame 21, a connecting conduit 24, a channel inlet/outlet conduit 22, the washing solution injection pump 9 is used for introducing washing solution into the sample extraction module 1 to purify the nucleic acid sample combined with the magnetic beads, and the fixing frame 21 is used for fixing a magnet on the side to avoid the influence of a strong magnetic field on the surrounding environment, so as to ensure safety; one end of the channel inlet and outlet conduit 22 is respectively communicated with the inlet 13 and the outlet 14 of the extraction module 1, and the other end is respectively communicated with the first multi-way pipe 4 and the second multi-way pipe 5, so that liquid leakage is avoided. And multi-way valves are arranged on the first multi-way pipe 4 and the second multi-way pipe 5 and are used for controlling the on-off of each guide pipe of the multi-way pipes.
Still be equipped with control module 3 on the extraction element, including voltage stabilizing circuit, touch screen display control circuit, microprocessor, two multi-ported valve drive circuit, a plurality of pump drive circuit and temperature control circuit etc.. The microprocessor can adopt a single chip microcomputer or an ARM processor, issues instructions to control the driving circuit, and further controls the corresponding valve, the pump and the heating module; the touch screen display controller receives control of the microprocessor and provides a display function for a user on one hand, and transmits a user instruction to the microprocessor through user touch screen feedback on the other hand; the voltage stabilizing circuit provides stable voltage for the microprocessor, the touch screen display controller and the driving circuit so as to keep the system stable.
The technical scheme of the invention provides an effective, rapid and semi-automatic method and device for extracting nucleic acid from a large-volume biological sample, the method can realize rapid extraction of nucleic acid from a sample of more than 10 ml, the extraction time is shorter than that of the traditional mode, the whole extraction link can be operated automatically, and the device can be combined with other detection devices in the later stage to achieve the purpose of full-automatic detection.
It should be understood that the above-described embodiments of the present invention are examples for clearly illustrating the invention, and are not to be construed as limiting the embodiments of the present invention, and it will be obvious to those skilled in the art that various changes and modifications can be made on the basis of the above description, and it is not intended to exhaust all embodiments, and obvious changes and modifications can be made on the basis of the technical solutions of the present invention.
Claims (10)
1. A nucleic acid extraction apparatus, comprising:
the sample extraction module is used for containing a nucleic acid sample and extracting nucleic acid from the sample and comprises an inlet, an outlet, a shell and an internal channel;
the magnetic bead injection pump is used for introducing a solution mixed with micron magnetic beads into the sample extraction module;
the magnetic field generating module is in a hollow arc shape and is used for generating a high-strength magnetic field;
a sample lysis module for separating nucleic acid from a sample and binding the nucleic acid to magnetic beads;
the eluent injection pump is used for introducing eluent into the sample extraction module to separate the nucleic acid from the magnetic beads to obtain nucleic acid;
the internal channel of the extraction module is consistent with the arc magnetic field constructed by the magnetic field generation module, so that the same or similar magnetic field is kept in each area in the channel, and magnetic beads are uniformly linked in the channel.
2. The nucleic acid extraction device of claim 1, wherein the sample extraction module is circular, the internal channels are uniformly arranged in the housing in an S-shape, and both ends of the internal channels are respectively connected with the inlet and the outlet.
3. The nucleic acid extraction device according to claim 2, wherein the magnetic field generation module comprises a large magnet, a small magnet and a base, the large magnet and the small magnet are arranged in the base in a circular arc shape and are concentrically arranged, the sample extraction module is arranged between the large magnet and the small magnet, and a high-intensity magnetic field is generated between the large magnet and the small magnet.
4. The nucleic acid extraction device according to claim 2, wherein the sample cracking module comprises a cracking channel tube, a heating tube, a heat insulation tube and a temperature probe, the cracking channel tube is connected to the inlet of the sample extraction module, the heating tube is sleeved on the outer surface of the cracking channel tube and heats the cracking channel tube, the heat insulation tube is sleeved on the outer surface of the heating tube and is used for reducing heat emission, and the temperature probe is connected to the cracking channel tube and is used for measuring the temperature of the cracking channel tube and feeding back the temperature.
5. The nucleic acid extraction device of claim 2, further comprising a washing solution injection pump, a first multi-way tube, a second multi-way tube, and a plurality of multi-way valves, wherein the washing solution injection pump is configured to introduce a washing solution into the sample extraction module to purify the nucleic acid sample bound to the magnetic beads; one end of the first multi-way tube is communicated with the inlet of the sample extraction module, and the other end of the first multi-way tube is respectively connected with the magnetic bead injection pump, the sample cracking module, the washing solution injection pump and the eluent injection pump; one end of the second multi-way pipe is communicated with the outlet of the sample extraction module, and the other end of the second multi-way pipe is respectively communicated with the extract and the waste liquid recovery unit.
6. The nucleic acid extraction device according to claim 5, wherein the extraction device further comprises a control module, and the control module comprises a control circuit and a microprocessor for controlling the operation state of each module in the extraction device and the opening and closing state of the multi-way valve.
7. A method for extracting nucleic acid using the nucleic acid extraction apparatus according to any one of claims 1 to 6, comprising:
introducing the solution mixed with the micron magnetic beads into a container, and placing the container in a magnetic field to enable the magnetic beads in the solution to be combined in a chain shape and fill the container;
cracking the sample added with the cracking liquid, and then introducing the sample into the container, so that the nucleic acid in the cracked sample is combined with the magnetic beads in the container and adsorbed in the container, and other impurities flow out of the outlet of the container along with the mixed solution;
introducing a washing solution into the container, and purifying the nucleic acid sample combined with the magnetic beads in the container;
and introducing an eluent into the container to separate the nucleic acid in the nucleic acid sample in the container from the magnetic beads.
8. The method for extracting nucleic acid according to claim 7, wherein the step of introducing the sample added with the lysis solution into the container after the lysis step comprises: and (3) cracking the sample added with the cracking solution for 10 minutes, and then introducing the sample into the container for 3-5 minutes.
9. The method for extracting nucleic acid according to claim 7, wherein a washing solution is mixed with the nucleic acid sample and kept for 3 to 5 minutes after the washing solution is introduced into the container.
10. The method for extracting nucleic acid according to claim 7, wherein an eluent is mixed with the nucleic acid sample and kept for 3 to 5 minutes after passing the eluent into the container.
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| CN109355283B (en) * | 2018-11-27 | 2023-05-05 | 中国科学院上海技术物理研究所 | Automatic nucleic acid extraction device suitable for space |
| CN109679832A (en) * | 2019-01-22 | 2019-04-26 | 周鸿坡 | A kind of high throughput automated magnetic beads for purifying instrument and its working method |
| CN113646419A (en) * | 2019-02-14 | 2021-11-12 | 高丽大学校算学协力团 | Extraction device for extracting target substance, extraction method, and fluidic chip |
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| CN112126642A (en) * | 2019-06-25 | 2020-12-25 | 京东方科技集团股份有限公司 | Nucleic acid extraction device and method thereof |
| CN110819623A (en) * | 2019-11-08 | 2020-02-21 | 珠海迪尔生物工程有限公司 | Nucleic acid extraction instrument and extraction method |
| CN111440703A (en) * | 2020-05-19 | 2020-07-24 | 广州高盛生物科技股份有限公司 | Double-extraction-mode nucleic acid extraction workstation and nucleic acid extraction method |
| US20220333048A1 (en) * | 2020-09-22 | 2022-10-20 | Boe Technology Group Co., Ltd. | Nucleic acid extraction microfluidic chip, and nucleic acid extraction device and extraction method |
| CN114763545A (en) * | 2021-01-12 | 2022-07-19 | 厦门致善生物科技股份有限公司 | Device, kit and method for extracting nucleic acid |
| CN112725162B (en) * | 2021-01-15 | 2022-03-01 | 山东大学 | Full-automatic microbial nucleic acid extraction and detection device and extraction and detection method |
| CN117070326A (en) * | 2023-10-19 | 2023-11-17 | 吉林大学 | Nucleic acid extraction device and extraction method thereof |
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