Negative-pressure nucleic acid extraction device with high-flux adsorption column

Abstract

The utility model provides a negative-pressure nucleic acid extraction device with a high-flux adsorption column, which solves the problems that the conventional centrifugal elution nucleic acid acquisition mode is more complicated to operate and samples are easy to mix, and adopts the main scheme that the negative-pressure nucleic acid extraction device comprises a liquid adding reagent tank, a liquid adding port and a liquid storage tank, the bottom of the liquid adding opening is communicated with a plurality of reagent drainage tubes which are linearly arranged at equal intervals; the U-shaped tube assembly is fixed at the inner bottom of the liquid adding reagent tank, one end of the U-shaped tube assembly is exposed out of the liquid adding reagent tank and is fixedly provided with a valve, a plurality of nucleic acid adsorption columns are arranged at the top of the U-shaped tube assembly at equal intervals in the axial direction, and the tops of the nucleic acid adsorption columns are communicated with the reagent drainage tubes in a one-to-one correspondence manner; the bottoms of the U-shaped pipe assemblies are communicated with the U-shaped pipe assemblies one by one through adapters; one end of the waste liquid bottle is communicated with the valve through a silicone tube, the other end of the waste liquid bottle is communicated with a vacuum controller, and the vacuum controller is used for carrying out negative pressure evacuation on the interior of the U-shaped tube assembly.

Description

Technical Field

[0001] The utility model relates to the technical field of nucleic acid extraction equipment, in particular to a high-throughput adsorption column negative pressure nucleic acid extraction device. Background Art

[0002] In the field of molecular biology research, it is often necessary to separate and purify macromolecules such as DNA and RNA in biological samples. Therefore, rapid separation and purification of DNA and RNA nucleic acid substances are key technologies to improve the efficiency of molecular biology research. Using silica purification columns to extract nucleic acid macromolecules has become a common method in molecular biology research. For silica column nucleic acid extraction technology, two common methods are: centrifugal separation and negative pressure air extraction separation method. The centrifugal method is a commonly used technique in laboratories currently. The specific operation process is as follows: After the cell sample is lysed with a lysis solution, centrifuge to obtain the supernatant; Add a nucleic acid column binding solution with a certain proportion of volume to the supernatant, mix and transfer it into the purification column; Place the purification column in a matching centrifuge tube and centrifuge; Take out the purification column and discard the filtrate in the centrifuge tube; Put the purification column back into the original centrifuge tube, add a nucleic acid washing solution, and centrifuge to remove the filtrate (repeat this step once); Finally, place the purification column in the centrifuge tube and spin it empty to remove the washing solution remaining on the purification column membrane; Place the purification column back into the centrifuge tube, add a certain amount of nucleic acid elution solution in the center of the purification column membrane, let it stand for a period of time, and then centrifuge for elution to obtain nucleic acid.

[0003] In the centrifugal method, the purification column needs to be used in cooperation with the centrifuge tube multiple times. For each centrifugation, the purification column needs to go through the process of being taken out from the centrifuge tube and reinserted. Therefore, the operation process is relatively complex, and it is easy to get confused when there are many samples, which is not conducive to processing multiple samples. For this reason, we propose a high-throughput adsorption column negative pressure nucleic acid extraction device to solve the above defects. Content of the Utility Model

[0004] The technical problem to be solved by the utility model is to overcome the defects existing in the prior art. The utility model proposes a high-throughput adsorption column negative pressure nucleic acid extraction device, which can not only continuously separate and purify samples, realize the advantages of negative pressure nucleic acid extraction, but also has simple operation, is easy to disassemble, has low maintenance cost and high use efficiency, and reduces the nucleic acid extraction time and cumbersome operation process.

[0005] To solve the above technical problem, the technical solution adopted by the utility model is: A high-throughput adsorption column negative pressure nucleic acid extraction device, comprising:

[0006] A liquid adding reagent tank, with a liquid adding port recessed at the top and hermetically sealed on one side through a reagent tank switch, and a plurality of linearly and equally spaced reagent drainage tubes communicating with the bottom of the liquid adding port;

[0007] A U-shaped tube assembly is fixed to the inner bottom of the liquid adding reagent tank, one end of which is exposed to the liquid adding reagent tank and fixed with a valve. The top of the U-shaped tube assembly is equidistantly arranged along its axial direction with multiple nucleic acid adsorption columns. The tops of the multiple nucleic acid adsorption columns are connected to the multiple reagent drainage tubes in a one-to-one correspondence, and the bottoms of the multiple nucleic acid adsorption columns are connected to the U-shaped tube assembly one by one through adapters.

[0008] The waste liquid bottle has one end connected to the valve through a silicone tube and the other end connected to a vacuum controller.

[0009] A vacuum controller is used to evacuate the U-shaped tube assembly under negative pressure.

[0010] Furthermore, an anti-backflow device is provided between the waste liquid bottle and the vacuum controller, and the anti-backflow device includes an overflow bottle, a liquid inlet hose and an air exhaust hose. The overflow bottle is transparent, the top of which is sealed and one end is connected to the waste liquid bottle through the liquid inlet hose, and the other end is connected to the vacuum controller through the air exhaust hose. The liquid inlet hose and the air exhaust hose are both inserted into the overflow bottle, and the bottom end of the air exhaust hose is higher than the bottom end of the liquid inlet hose.

[0011] Furthermore, the top of the waste liquid bottle is sealed and pre-opened with an air inlet and an air outlet, the air inlet is correspondingly connected to the silicone tube, and the air outlet is correspondingly connected to the liquid inlet hose.

[0012] Furthermore, the nucleic acid adsorption column is made of PE plastic, has a cylindrical structure, a wall thickness of 2 mm, a diameter of 20 mm, and a height of 100 mm, and the adsorption membrane built into the nucleic acid adsorption column is a silica fiber membrane.

[0013] Furthermore, the U-shaped tube assembly and the liquid adding reagent tank are both made of stainless steel, and the surfaces are treated for corrosion resistance. The size of the U-shaped tube is 300*200*80mm, and the liquid adding reagent tank is a rectangular structure with corresponding dimensions of 380*280*400mm. The size of the liquid adding port is 300*200*50mm.

[0014] Furthermore, the number of the reagent drainage tubes is not less than 24.

[0015] Furthermore, the vacuum controller is a circulating water vacuum pump or an oil pressure vacuum pump, and its negative pressure can reach -0.09 MPa under relatively sealed working conditions.

[0016] Compared with the prior art, the beneficial effects of the present invention include:

[0017] 1. The use of negative pressure pumping replaces the complicated centrifugation process in most centrifugation methods, making the entire operation process time-saving and labor-saving, and capable of continuously separating and purifying multiple samples;

[0018] 2. Simple operation, easy disassembly, low maintenance cost, high usage efficiency, reducing nucleic acid extraction time and cumbersome operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The disclosure of the present utility model will be described with reference to the accompanying drawings. It should be understood that the drawings are only for illustrative purposes and are not intended to limit the scope of protection of the present utility model. In the drawings, the same reference numerals are used to refer to the same components. Among them:

[0020] Figure 1 Schematically shows a schematic diagram of the overall structure according to an embodiment of the present utility model;

[0021] Figure 2 Schematically shows a schematic diagram of the adapter structure according to an embodiment of the present utility model.

[0022] Reference numerals in the figure: 1, liquid addition reagent tank; 2, nucleic acid adsorption column; 3, adapter; 4, U-shaped tube assembly; 5, reagent drainage tube; 6, reagent tank switch; 7, waste liquid bottle; 8, silica gel tube; 9, anti-backflow device; 10, vacuum controller; 11, air inlet; 12, air outlet; 13, liquid addition port; 14, overflow bottle; 15, liquid inlet hose; 16, air extraction hose. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] It is easy to understand that according to the technical solution of the present utility model, without changing the essential spirit of the present utility model, those of ordinary skill in the art can propose various interchangeable structural ways and implementation ways. Therefore, the following detailed embodiments and the accompanying drawings are only exemplary descriptions of the technical solution of the present utility model and should not be regarded as all of the present utility model or as a limitation or restriction on the technical solution of the present utility model.

[0024] According to an embodiment of the present utility model in combination with Figure 1 - Figure 2 shown.

[0025] For the overall structure, in this embodiment, a high-throughput adsorption column negative pressure nucleic acid extraction device includes:

[0026] A liquid addition reagent tank 1, with a liquid addition port 13 recessed at the top and movably sealed on one side by a reagent tank switch 6, and a plurality of reagent drainage tubes 5 arranged linearly and equidistantly are connected to the bottom of the liquid addition port 13;

[0027] The U-shaped tube assembly 4 is fixed to the inner bottom of the liquid adding reagent tank 1. One end of the U-shaped tube assembly 4 is exposed outside the liquid adding reagent tank 1 and is fixed with a valve. At the top of the U-shaped tube assembly 4, a plurality of nucleic acid adsorption columns 2 are arranged at equal intervals along its axis. The tops of the plurality of nucleic acid adsorption columns 2 are in one-to-one correspondence and communication with a plurality of reagent drainage tubes 5, and their bottoms are connected to the U-shaped tube assembly 4 through adapters 3 one by one.

[0028] The waste liquid bottle 7, one end of which is connected to the valve through a silica gel tube 8, and the other end is connected to a vacuum controller 10.

[0029] The vacuum controller 10 is used to evacuate the inside of the U-shaped tube assembly 4 to a negative pressure.

[0030] Furthermore, in the present application, an anti-backflow device 9 is further provided between the waste liquid bottle 7 and the vacuum controller 10. The anti-backflow device 9 includes an overflow bottle 14, a liquid inlet hose 15, and an air extraction hose 16. The overflow bottle 14 is transparent, its top is sealed, and one end is connected to the waste liquid bottle 7 through the liquid inlet hose 15, and the other end is connected to the vacuum controller 10 through the air extraction hose 16. The liquid inlet hose 15 and the air extraction hose 16 are both inserted into the overflow bottle 14, and the bottom end height of the air extraction hose 16 is higher than the bottom end height of the liquid inlet hose 15. The top of the waste liquid bottle 7 is sealed and is pre-opened with an air inlet 11 and an air outlet 12. The air inlet 11 is correspondingly connected to the silica gel tube, and the air outlet 12 is correspondingly connected to the liquid inlet hose.

[0031] For the specific size selection of each structural member, in this embodiment, the nucleic acid adsorption column 2 is made of PE plastic, which is a cylindrical structure with a wall thickness of 2 mm, a diameter of 20 mm, and a height of 100 mm. The adsorption membrane built in the nucleic acid adsorption column 2 is a silica gel fiber membrane. The U-shaped tube assembly 4 and the liquid adding reagent tank 1 are both made of stainless steel and their surfaces are both treated with corrosion resistance. The size of the U-shaped tube is 300*200*80 mm. The liquid adding reagent tank 1 is a cuboid structure with corresponding dimensions of 380*280*400 mm. The size of the liquid adding port 13 is 300*200*50 mm. The reagent drainage tubes 5 are not less than 24. The vacuum controller 10 is a circulating water vacuum pump or an oil pressure vacuum pump, and the negative pressure under its working relatively sealed condition can reach -0.09 Mpa.

[0032] Through the above structure, the upper part of the U-shaped tube assembly 4 is a sample receiving port, and the lower part is a liquid discharging port. At the sample receiving port, it is connected to the nucleic acid adsorption column 2 through an adapter 3, and at the liquid discharging port, it is connected to the air inlet 11 of the waste liquid collection bottle through a silica gel tube; the air outlet 12 of the waste liquid collection bottle is also connected to the negative pressure controller with a silica gel tube 8. To prevent liquid backflow, an anti-backflow device 9 is connected in series between the waste liquid collection bottle and the negative pressure controller.

[0033] The nucleic acid adsorption column 2 can be used to extract nucleic acids, such as DNA or RNA, and can also be used to remove impurities during the pretreatment of biological samples. Its material is medical-grade polypropylene, and the production process is free from nucleic acid and nuclease contamination. At the same time, the silica gel membrane used in the adsorption column is of a general-purpose specification on the market. The purification principle of this silica gel membrane for nucleic acids is: adsorption with high salt and elution with low salt.

[0034] The specific operation process of the negative pressure method is as follows: Insert the nucleic acid adsorption column 2 onto the adapter 3 of the U-shaped tube assembly 4; lyse the cell sample and take the supernatant, and add a certain proportion of the nucleic acid column binding solution by volume through the liquid addition port 13 of the liquid addition reagent tank 1 to the supernatant, and transfer the mixture to the adsorption column after mixing.

[0035] After the nucleic acid is adsorbed into the nucleic acid adsorption column 2, turn on the negative pressure controller to suck the waste liquid in the nucleic acid adsorption column 2 into the waste liquid bottle 7, and observe whether there is any waste liquid leakage from the bottom end of the liquid inlet hose 15 in the overflow bottle 14. After the nucleic acid extraction is completed, the negative pressure can be maintained, and the adsorption column can be washed with the washing solution. Under the negative pressure state, the residual washing solution on the adsorption column membrane is removed; place the adsorption column in a new centrifuge tube, add a certain amount of nucleic acid elution solution to the center of the adsorption column membrane, let it stand for a period of time, and then centrifuge and elute to obtain the nucleic acid. The advantage of the negative pressure method is that it replaces most of the complicated centrifugation processes in the centrifugation method by using the negative pressure pumping method, making the whole operation process time-saving and labor-saving, and capable of continuously separating and purifying multiple samples.

[0036] The high-throughput adsorption column negative pressure nucleic acid extraction device of the present utility model can be used to extract nucleic acids by means of continuous negative pressure pumping. The following is a further description in combination with the specific extraction process.

[0037] For the large-scale extraction of high-purity plasmid DNA from multiple samples:

[0038] 1. Consumables and reagents

[0039] A centrifuge and 50-ml centrifuge tubes, solutions P1, P2, P4 for plasmid lysis, nuclease RNase A, binding promoting solution, washing solution, elution solution.

[0040] 2. Steps

[0041] Twenty-four different strains of Escherichia coli containing high-copy-number plasmids (the plasmids are ampicillin-resistant) were inoculated into 100 ml of TB medium with a final concentration of 100 ng / ml of ampicillin and cultured for 16 hours. They were collected into centrifuge tubes and centrifuged at 4200 rpm at room temperature for 5 minutes, and the bacterial cell pellets were collected separately. Carefully remove the supernatant, add 10 ml of P1 (check whether RNase A is added before using P1), vortex or pipette to mix the bacterial cells evenly; then add 10 ml of P2, gently mix by inverting back and forth 10 - 15 times to obtain a clear lysate; then add 10 ml of P4, invert and mix several times until a uniform white flocculent precipitate appears, and let it stand at room temperature for 2 minutes to fully neutralize; centrifuge at 4200 rpm at room temperature for 5 minutes to obtain the supernatant; transfer the supernatant obtained in the previous step to the binding solution, shake well; then pour the well-shaken supernatant binding solution mixture into the prepared nucleic acid adsorption column 2 (the adsorption column is pre-equilibrated with PS) and make marks; insert the adsorption columns together with the U-shaped tube assembly 4 through the adapter 3 in the order of the marks, connect all components, turn on the vacuum controller 10, suck out the liquid in the nucleic acid adsorption column 2, and turn off the vacuum controller 10 when the liquid is completely sucked out; open the liquid addition reagent tank 1, let the washing reagent flow into the nucleic acid adsorption column 2 through the reagent drainage tube 5 of the liquid addition reagent tank 1, about 10 ml, turn on the vacuum controller 10 until the detergent in the adsorption column is sucked out (this step is repeated once); maintain negative pressure for 10 minutes to remove the residual washing liquid on the membrane of the nucleic acid adsorption column 2; remove the nucleic acid adsorption column 2 and put it into a new 50 ml centrifuge tube, add 1.5 ml of elution buffer, let it stand at room temperature for 3 - 5 minutes, and centrifuge at 4200 rpm for 10 minutes to elute the plasmid DNA.

[0042] 3. Plasmid quality detection

[0043] The quality of the extracted plasmid was detected using agarose gel electrophoresis and a nucleic acid concentration detector. The detection results showed that the concentration and purity of the isolated and purified plasmid reached the experimental requirements; the quality parameter ranges of the 24 samples were: plasmid concentration 500 - 1200 ng / μl, A260 / 280 range 1.82 - 1.96, A260 / 230 range 1.95 - 2.60.

[0044] The above extraction process is a common application of the high-throughput adsorption column negative pressure nucleic acid extraction device. The use of this device greatly improves the efficiency of extracting a large number of plasmids; the time for this device to isolate and purify 24 samples is only 1 / 3 of the time for the centrifugation method to extract 24 samples. At the same time, for the treatment of less than 24 samples, this device can also be applied. The specific operation is to block the unused holes of the U-shaped tube assembly 4 with rubber stoppers.

[0045] The technical scope of the present utility model is not limited to the content described above. Those skilled in the art can make various deformations and modifications to the above embodiments without departing from the technical idea of the present utility model, and these deformations and modifications shall fall within the protection scope of the present utility model.

Claims

1. A high-throughput adsorption column negative pressure nucleic acid extraction device, characterized in that, include: A liquid adding reagent tank, with a liquid adding port recessed on the top and movably sealed on one side by a reagent tank switch, and a plurality of linearly equidistantly arranged reagent drainage tubes connected to the bottom of the liquid adding port; A U-shaped tube assembly is fixed to the inner bottom of the liquid adding reagent tank, one end of which is exposed to the liquid adding reagent tank and fixed with a valve. The top of the U-shaped tube assembly is equidistantly arranged along its axial direction with multiple nucleic acid adsorption columns. The tops of the multiple nucleic acid adsorption columns are connected to the multiple reagent drainage tubes in a one-to-one correspondence, and the bottoms of the multiple nucleic acid adsorption columns are connected to the U-shaped tube assembly one by one through adapters. The waste liquid bottle has one end connected to the valve through a silicone tube and the other end connected to a vacuum controller. A vacuum controller is used to evacuate the U-shaped tube assembly under negative pressure.

2. The high-throughput adsorption column negative pressure nucleic acid extraction device according to claim 1, wherein: An anti-backflow device is also provided between the waste liquid bottle and the vacuum controller, and the anti-backflow device includes an overflow bottle, a liquid inlet hose and an air extraction hose. The overflow bottle is transparent, with a sealed top and one end connected to the waste liquid bottle through the liquid inlet hose, and the other end connected to the vacuum controller through the air extraction hose. The liquid inlet hose and the air extraction hose are both inserted into the overflow bottle, and the bottom end of the air extraction hose is higher than the bottom end of the liquid inlet hose.

3. The high-throughput adsorption column negative pressure nucleic acid extraction device according to claim 2, wherein: The top of the waste liquid bottle is sealed and pre-opened with an air inlet and an air outlet, the air inlet is correspondingly connected to the silicone tube, and the air outlet is correspondingly connected to the liquid inlet hose.

4. The high-throughput adsorption column negative pressure nucleic acid extraction device according to claim 1, characterized in that: The nucleic acid adsorption column is made of PE plastic, has a cylindrical structure, a wall thickness of 2 mm, a diameter of 20 mm, and a height of 100 mm, and the adsorption membrane built into the nucleic acid adsorption column is a silica fiber membrane.

5. The high-throughput adsorption column negative pressure nucleic acid extraction device according to claim 1, characterized in that: The U-shaped tube assembly and the liquid adding reagent tank are both made of stainless steel, and the surface has been treated for corrosion resistance. The size of the U-shaped tube is 300*200*80mm, and the liquid adding reagent tank is a rectangular structure with corresponding dimensions of 380*280*400mm. The size of the liquid adding port is 300*200*50mm.

6. The high-throughput adsorption column negative-pressure nucleic acid extraction device according to claim 1, wherein: The number of the reagent drainage tubes is no less than 24.

7. The high-throughput adsorption column negative pressure nucleic acid extraction device according to claim 1, characterized in that: The vacuum controller is a circulating water vacuum pump or an oil pressure vacuum pump, and its negative pressure can reach -0.09 MPa under relatively sealed working conditions.

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