CN116355724A - Nucleic acid extraction device and nucleic acid extraction method - Google Patents
Nucleic acid extraction device and nucleic acid extraction method Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims abstract description 100
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 99
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 99
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 99
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 122
- 239000007788 liquid Substances 0.000 claims abstract description 59
- 230000033444 hydroxylation Effects 0.000 claims abstract description 20
- 238000005805 hydroxylation reaction Methods 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000000945 filler Substances 0.000 claims abstract description 4
- 239000004005 microsphere Substances 0.000 claims description 44
- 239000000377 silicon dioxide Substances 0.000 claims description 23
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 22
- 235000012239 silicon dioxide Nutrition 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 13
- 238000007599 discharging Methods 0.000 claims description 11
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- 239000003480 eluent Substances 0.000 claims description 10
- 230000005284 excitation Effects 0.000 claims description 10
- 239000002699 waste material Substances 0.000 claims description 8
- 239000003792 electrolyte Substances 0.000 claims description 7
- 238000005530 etching Methods 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 230000009089 cytolysis Effects 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 15
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 238000010521 absorption reaction Methods 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 26
- 238000011049 filling Methods 0.000 description 15
- 239000013592 cell lysate Substances 0.000 description 9
- 239000011324 bead Substances 0.000 description 8
- 229910052710 silicon Inorganic materials 0.000 description 8
- 239000010703 silicon Substances 0.000 description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 7
- 238000000926 separation method Methods 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000003746 surface roughness Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 229910002808 Si–O–Si Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
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- 238000010828 elution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
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- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- 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
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Abstract
The present application relates to a nucleic acid extraction device and a nucleic acid extraction method, and relates to the field of biomedical detection technology, the nucleic acid extraction device includes: an extraction tube having a fill port and a drain port; a filter screen is arranged at one end, close to the liquid outlet, of the extraction pipe, a core rod is movably connected at one end, close to the filler outlet, of the extraction pipe, and liquid in the extraction pipe is forced to be discharged from the liquid outlet through the core rod; silica microspheres are filled between the filter screen and the core rod in the extraction tube, and the silica microspheres are modified silica microspheres subjected to surface hydroxylation treatment. The extraction of nucleic acid in this application is only through the liquid removal in the movable core pole promotion extraction tube, and the absorption effect through modified silica microballon self in the liquid removal in-process adsorbs the extraction nucleic acid, does not need high-speed centrifuge's centrifugal processing, and operating mode is simple high-efficient, and operating time is short, low cost.
Description
Technical Field
The present application relates to the field of biomedical detection technology, and in particular, to a nucleic acid extraction device and a nucleic acid extraction method.
Background
Nucleic acids (including DNA and RNA) are commonly used as raw materials for various analyses and experiments in medicine, pharmaceutical research, and clinical diagnostics. Currently, a sufficient amount of nucleic acid is readily available by a variety of automated in vitro nucleic acid amplification techniques, such as the well-known Polymerase Chain Reaction (PCR) techniques, but extraction or purification of the nucleic acid is often required prior to amplification.
The main principal of the currently mainstream nucleic acid extraction methods is a column method and a magnetic bead method, wherein the basic principle is that a lysis solution is utilized to promote cell disruption, so that nucleic acid in the cells is released, then the released nucleic acid is specifically adsorbed on a centrifugal column or magnetic beads, and the materials of the centrifugal column and the magnetic beads support the nucleic acid with stronger affinity and adsorption force and basically do not adsorb other biochemical components in the disrupted cell solution, such as proteins, polysaccharides, lipids and the like, so that the separation and extraction of the nucleic acid can be completed; after the nucleic acid is adsorbed on the centrifugal column or the magnetic beads, the centrifugal column or the magnetic beads are rinsed by the rinsing liquid to remove impurities, and finally the nucleic acid is eluted by the eluent to obtain purified nucleic acid for subsequent nucleic acid amplification detection.
The current mainstream column method and magnetic bead method are used for extracting nucleic acid, the operation process is complicated, the operation time is long, the nucleic acid extraction cost is high, and the method is difficult to widely popularize and use in emergency rooms and hospitals with relatively poor experimental conditions.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a nucleic acid extraction device and a nucleic acid extraction method, so as to simplify the nucleic acid extraction process, reduce the nucleic acid extraction cost and improve the efficiency.
In a first aspect, the present application provides a nucleic acid extraction apparatus, which adopts the following technical scheme:
a nucleic acid extraction device comprising: an extraction tube having a fill port and a drain port; a filter screen is arranged at one end, close to the liquid outlet, of the extraction pipe; a core rod is movably connected to one end of the extraction pipe close to the filling port, and liquid in the extraction pipe is forced to be discharged from the liquid discharge port through the core rod; and a silicon dioxide microsphere forming extraction layer is filled between the filter screen and the core rod in the extraction pipe, and the silicon dioxide microsphere is a modified silicon dioxide microsphere subjected to surface hydroxylation treatment.
Through the adoption of the technical scheme, the surface hydroxyl-treated modified silicon dioxide microspheres are obviously improved in the quantity of exposed hydroxyl groups on the surfaces, and electronegativity of the silicon hydroxyl groups can induce cations in a high-salt solution (pyrolysis liquid) to be transferred into a water molecule layer to form an electropositive salt bridge structure, so that nucleic acid with electronegative phosphate frameworks can be rapidly captured. Filling the modified silica microspheres in an extraction tube, adsorbing nucleic acid in a cell lysate by the modified silica microspheres when the cell lysate flows through a filling area of the modified silica microspheres under the pushing action of a core rod, discharging waste liquid from a liquid outlet through a filter screen, adsorbing the nucleic acid on the surface of the modified silica microspheres and trapping the nucleic acid and the silica microspheres in the extraction tube, and sequentially carrying out impurity removal of a rinsing liquid and elution treatment of an eluent to obtain purified nucleic acid. In the device, the extraction of the nucleic acid only pushes the liquid in the extraction tube to move through the movable core rod, the nucleic acid is extracted by the self adsorption effect of the modified silica microspheres in the liquid moving process, the filling amount of the modified silica can be adjusted, the centrifugal treatment of a high-speed centrifugal machine is not needed, the device can be suitable for a non-uniform system, the good separation effect can be achieved in a liquid-phase non-uniform system, the operation mode is simple and efficient, the operation time is short, the cost is low, and the device can be widely applied to the rapid diagnosis of the nucleic acid in a basic-level hospital and an emergency room with relatively poor experimental conditions.
Optionally, the modified silica microspheres are prepared by the following method:
placing the silicon dioxide microspheres in a sodium carbonate solution, and heating to 80-90 ℃ to perform surface etching; and (3) after the surface etching is finished, carrying out electric excitation hydroxylation treatment on the silica microspheres in magnesium chloride electrolyte, and obtaining the surface modified silica microspheres after the electric excitation hydroxylation treatment is finished.
Optionally, the mass concentration of the sodium carbonate solution is 30-40 wt%.
Optionally, the mass fraction of the magnesium chloride electrolyte is 0.02-0.025%.
Optionally, the electro-active hydroxylation treatment is direct current electro-active, the voltage is 1.5-1.7V/cm, and the electro-active hydroxylation treatment time is 1-2 h.
By adopting the technical scheme, the surface of the silicon dioxide microsphere is etched through the sodium carbonate solution to increase the surface roughness, and then the electric excitation hydroxylation treatment is carried out, so that the silicon dioxide surface promotes the Si-O-Si bond to break bond and react with H in water in the direct current electric excitation process + 、OH - And combining, increasing the number of silicon hydroxyl groups on the surface of the silicon dioxide to obtain the surface modified silicon dioxide microsphere. After surface hydroxylation modification treatment, the surface hydroxyl content of the silicon dioxide microsphere can reach (7.5+/-1.3) OH/nm 2 . The silicon dioxide microsphere is subjected to modification to expose more hydroxyl groups, and the electronegativity of the silicon hydroxyl groups can induce cations in the lysate to be transferred into a water molecule layer to form a positive salt bridge structure, so that nucleic acid with electronegative phosphate skeleton is rapidly captured, and the effect of extracting the nucleic acid is achieved. Compared with a centrifugal column or magnetic beads, the modified silica microsphere has good adsorption effect on nucleic acid, has a simple treatment mode and low cost, and can achieve good nucleic acid separation effect in a liquid phase heterogeneous system.
Optionally, the silica microspheres are micron-sized silica microspheres.
By adopting the technical scheme, the micron-sized silica microspheres have large specific surface area, if the granularity of the silica microspheres is too small, the specification requirement on a filter screen is obviously increased, the cost is increased, and the discharge of impurities in the cell lysate is not facilitated; when the particle size of the silica microsphere is too large, the specific surface area of the silica microsphere is reduced, so that the number of bare silicon hydroxyl groups in the whole filling area is reduced, gaps among the silica microsphere particles are enlarged, and the nucleic acid adsorption effect is affected.
Optionally, the core rod is in threaded connection or sliding connection with the extraction tube.
By adopting the technical scheme, a structure similar to an injector is formed between the core rod and the extraction tube, the pressure in the extraction tube is increased by pushing the core rod or rotating the core rod, so that the cell lysate in the extraction tube is forced to flow through the modified silica microsphere filling area, and the nucleic acid in the cell lysate is adsorbed on the surface of the modified silica microsphere, so that the separation of the nucleic acid and other components in the cell lysate is completed.
In a second aspect, the present application provides a method for extracting nucleic acid, which adopts the following technical scheme:
a method for extracting nucleic acid, comprising the steps of:
s1, mixing a nucleic acid sample with a lysis solution to fully lyse the sample;
s2, adding the mixed solution obtained in the step S1 into an extraction tube of the nucleic acid extraction device, uniformly mixing the modified silica microspheres, and discharging waste liquid from a liquid outlet;
s3, adding a rinsing liquid into the extraction tube, uniformly mixing the modified silicon dioxide microspheres, discharging waste liquid from a liquid outlet, and washing off impurities attached to the surface of the modified silicon dioxide;
s4, adding an eluent into the extraction tube, uniformly mixing the modified silicon dioxide microspheres, discharging the modified silicon dioxide microspheres from a liquid outlet, and collecting the eluent to obtain purified nucleic acid.
According to the technical scheme, when the nucleic acid extraction device is used for extracting nucleic acid through the method, modified silica microspheres are filled according to requirements, then cell lysate is injected into an extraction pipe from a filling port, a core rod is installed, liquid in the extraction pipe is forced to flow through a filling area of the modified silica microspheres through movement of the core rod and finally discharged from a liquid outlet, then rinsing liquid and eluent are sequentially filled, the microspheres are uniformly mixed after each filling, waste liquid is pumped out, and nucleic acid adsorbed on the surfaces of the modified silica microspheres is discharged after impurity removal, so that purified nucleic acid is obtained. In the nucleic acid extraction method, only cell lysate, rinsing liquid and eluent are sequentially filled into the extraction tube and are discharged, the operation of extracting the nucleic acid is completed in the liquid discharge process, the traditional high-speed centrifugation operation is not needed, the extraction mode is simple and efficient, the operation time is short, and the cost is low.
In summary, the present application includes at least one of the following beneficial technical effects:
1. the utility model provides a nucleic acid extraction device, the extraction of nucleic acid is only through the liquid removal in the movable core pole promotion extraction tube, adsorb extraction nucleic acid through the adsorption effect of modified silica microballon self in the liquid removal in-process, and the filling amount of modified silica can be adjusted, need not high-speed centrifuge's centrifugal processing, can be applicable to inhomogeneous system, also can reach fine separation effect in the inhomogeneous system of liquid phase, operating means is simple high-efficient, operating time is short, low cost, can wide application in the relatively worse basic unit hospital of experimental condition and the quick diagnosis of emergency room's nucleic acid.
2. The modified silica microsphere that this application provided is through carrying out surface hydroxylation to silica microsphere for silica surface exposure's hydroxyl quantity promotes by a wide margin, and the electronegativity of silicon hydroxyl can induce cation in the lysate to shift to the water molecule layer in form positive electric salt bridge structure, catches the nucleic acid that has electronegativity phosphoric acid skeleton fast, reaches the effect of nucleic acid extraction. Compared with a centrifugal column or magnetic beads, the modified silica microsphere has good adsorption effect on nucleic acid, has a simple treatment mode and low cost, and can achieve good nucleic acid separation effect in a liquid phase heterogeneous system.
3. The nucleic acid extraction method provided by the application depends on the nucleic acid extraction device in the technical scheme, can achieve simple and efficient nucleic acid extraction effect, has short operation time and low cost, and is suitable for large-scale popularization and application.
Drawings
FIG. 1 is a schematic diagram showing the structure of a nucleic acid isolation apparatus according to an embodiment of the present application.
Reference numerals illustrate: 1. an extraction tube; 11. a filler port; 12. a liquid outlet; 2. a core bar; 3. a filter screen; 4. and (5) extracting the layer.
Detailed Description
The present application is described in further detail below with reference to the accompanying drawings and specific examples.
Referring to fig. 1, the nucleic acid extraction device comprises an extraction tube 1 and a core bar 2 installed inside the extraction tube 1, wherein both ends of the extraction tube 1 are respectively provided with a filling port 11 and a liquid discharge port 12, the core bar 2 can move axially along the extraction tube 1 inside the extraction tube 1 and can be installed or removed from the filling port 11 of the extraction tube 1, and the outer diameter of one end, close to the liquid discharge port 12, of the core bar 2 is consistent with the inner diameter of the extraction tube 1, so that the air pressure inside the extraction tube 1 can be changed along with the movement of the core bar 2, and the purpose of discharging liquid in the extraction tube 1 from the liquid discharge port 12 is achieved; the core rod 2 and the extraction tube 1 can be in sliding connection or threaded connection. The filter screen 3 is fixedly arranged at the position, close to the liquid outlet 12, in the extraction pipe 1, a modified silicon dioxide microsphere is filled above the filter screen 3, the filled silicon dioxide modified microsphere forms an extraction layer 4, the modified silicon dioxide microsphere is filled between the filter screen and the core rod 2, the aperture of a water filtering hole on the filter screen 3 is smaller than the particle size of the modified silicon dioxide microsphere, and further, the liquid can be ensured to permeate through the filter screen 3 but the modified silicon dioxide microsphere is impermeable.
Preparation example of modified silica microsphere
The modified silica microspheres in the present application are prepared by the following method:
preparation example 1
The preparation method of the modified silicon dioxide microsphere comprises the following steps:
s1, placing micron-sized silicon dioxide microspheres in a reaction container, adding a sodium carbonate solution with the mass fraction of 30%, uniformly mixing, heating to 80 ℃ for surface etching, and increasing the surface roughness of the silicon dioxide microspheres for 1h;
s2, placing the silicon dioxide microspheres with the etched surfaces into an electrolytic cell for electric excitation hydroxylation treatment, wherein the electrolyte is magnesium chloride solution with the mass fraction of 0.025%, the voltage is 1.5V/cm, and the direct current is electrically excited for 1h to obtain the modified silicon dioxide microspheres.
Preparation example 2
The difference between this preparation example and preparation example 1 is that the mass fraction of the sodium carbonate solution was 40%, the DC excitation voltage was 1.7V/cm, and the mass fraction of the electrolyte was 0.02%. The remainder remained the same as in preparation example 1.
Preparation example 3
The difference between this preparation example and preparation example 1 is that the mass fraction of the sodium carbonate solution was 30%, the DC excitation voltage was 1.6V/cm, and the mass fraction of the electrolyte was 0.025%. The remainder remained the same as in preparation example 1.
Preparation example 4
The difference between this preparation example and preparation example 1 is that the silica microspheres were directly subjected to the electro-excitation treatment without sodium carbonate etching, and the remainder were the same as in preparation example 1.
Preparation example 5
The difference between this preparation and preparation 1 is that the silica microspheres were etched only with sodium carbonate solution, were not subjected to electrically stimulated hydroxylation, and remain the same as preparation 1.
Example 1
A method for extracting nucleic acid, comprising the steps of:
s1, mixing a nucleic acid sample with a lysis solution to fully lyse the sample; the lysate included 40mg/mL proteinase K,10mg/mL lysozyme, 6mol/L guanidine hydrochloride, 1mmol/L EDTA,33% (v/v) isopropanol, 0.1% SDS,0.5% Triton-100, 0.5% beta-mercaptoethanol, 800mmol/L sodium chloride, 200mmol/L sodium citrate (pH=6);
s2, adding the mixed solution into an extraction pipe, uniformly mixing the mixed solution with the modified silica microspheres in the extraction pipe, pushing the mixed solution to flow through a modified silica microsphere filling area by moving a core rod, and discharging waste liquid from a liquid outlet; the modified silica microspheres in this example were prepared from preparation example 1;
s3, adding a rinsing liquid into the extraction tube, uniformly mixing with the silicon dioxide microspheres, continuously pushing the mixed liquid to flow through a modified silicon dioxide microsphere filling area by moving the core rod, and discharging the waste liquid from the liquid outlet; the rinsing liquid comprises rinsing liquid A and rinsing liquid B, the rinsing liquid A and the rinsing liquid B are sequentially used for cleaning, the rinsing liquid A comprises 800mmol/L lithium chloride, 50mmol/L MOPS,100mmol/L sodium chloride and 60% ethanol (pH is 7.0); the rinsing liquid B is 70% ethanol;
s4, adding an eluent into the extraction tube, uniformly mixing with the silicon dioxide microspheres, pushing the mixed solution to flow through a modified silicon dioxide microsphere filling area by moving the core rod again, and discharging and collecting the eluent from a liquid outlet to obtain purified nucleic acid; the eluate included 0.5mmol/LEDTA,10mmol/L tris-HCl (ph=8.0).
Example 2
This example differs from example 1 in that modified silica microspheres were prepared from preparation 2, the remainder remaining in accordance with example 1.
Example 3
This example differs from example 1 in that modified silica microspheres were prepared from preparation 3, the remainder remaining in accordance with example 1.
Example 4
This example differs from example 1 in that modified silica microspheres were prepared from preparation 4, the remainder remaining in accordance with example 1.
Example 5
This example differs from example 1 in that modified silica microspheres were prepared from preparation 5, the remainder remaining in accordance with example 1.
Example 6
This example differs from example 1 in that the particle size of the silica microspheres is 500 to 800 μm, the remainder remaining the same as example 1.
Example 7
This example differs from example 1 in that the silica microspheres are nanoscale silica microspheres, the remainder remaining in accordance with example 1.
Comparative example 1
In this comparative example, a purified nucleic acid sample was obtained by extracting nucleic acid from a sample using a full-automatic nucleic acid extractor from Hangzhou ao Cheng Yiqi Co.
Comparative example 2
This example is different from example 1 in that the silica microspheres are silica microspheres which have not been subjected to surface hydroxylation treatment, and the remainder remains the same as in example 1.
Performance detection
1. Hydroxyl number on the surface of modified silica microsphere: the number of silicon hydroxyl groups on the surface of the modified silica microspheres prepared in preparation examples 1 to 5 was measured by a titration method, and silica microspheres which were not subjected to the indicated hydroxylation treatment were selected as a control group for measurement, and the measurement results are shown in Table 1 below.
Table 1: preparation examples 1 to 5 results of measuring the number of hydroxyl groups on the surface of silica microspheres
As can be seen from the data in table 1, the number of silicon hydroxyl groups on the surface of the silica microspheres increases significantly after the surface hydroxylation treatment. And the silicon hydroxyl on the surface of the silicon dioxide can be better exposed through the combination of sodium carbonate solution etching treatment and electric excitation hydroxylation treatment.
2. The extraction effect is as follows: the nucleic acid samples extracted in examples 1 to 7 and comparative examples 1 to 2 were subjected to nucleic acid concentration detection, and the detection results are shown in Table 2 below.
Table 2: results of concentration detection of nucleic acids of examples 1 to 7 and comparative examples 1 to 2
As can be seen from the data in Table 2, the nucleic acid extraction method provided by the application can effectively extract the nucleic acid in the cell lysate, and has high extraction efficiency and higher purity. The purified nucleic acid obtained by using the nucleic acid extraction device and the nucleic acid extraction method in the application is obviously different from the concentration and purity of the nucleic acid extracted by using a mature nucleic acid extraction instrument in the prior art, and the extraction method is simple, short in operation time and low in cost, and can be well applied to emergency departments or remote village and town hospitals for extracting the nucleic acid.
The foregoing are all preferred embodiments of the present application, and are not intended to limit the scope of the present application in any way, therefore: all equivalent changes in structure, shape and principle of this application should be covered in the protection scope of this application.
Claims (8)
1. A nucleic acid extraction device, comprising:
an extraction pipe (1) having a filler port (11) and a drain port (12);
a filter screen (3) is arranged at one end, close to a liquid outlet (12), of the extraction pipe (1), a core rod (2) is movably connected at one end, close to a filler port (11), of the extraction pipe (1), and liquid in the extraction pipe (1) is forced to be discharged from the liquid outlet (12) through the core rod (2);
the extraction pipe (1) is internally filled with a silicon dioxide microsphere forming extraction layer (4) between the filter screen (3) and the core rod (2), and the silicon dioxide microsphere is a modified silicon dioxide microsphere subjected to surface hydroxylation treatment.
2. The nucleic acid extraction device of claim 1, wherein the modified silica microspheres are prepared by: placing the silicon dioxide microspheres in a sodium carbonate solution, and heating to 80-90 ℃ to perform surface etching; and (3) after the surface etching is finished, carrying out electric excitation hydroxylation treatment on the silica microspheres in magnesium chloride electrolyte, and obtaining the surface modified silica microspheres after the electric excitation hydroxylation treatment is finished.
3. The nucleic acid isolation apparatus according to claim 2, wherein the sodium carbonate solution has a mass concentration of 30 to 40wt%.
4. The nucleic acid extraction apparatus according to claim 2, wherein the magnesium chloride electrolyte is 0.02 to 0.025% by mass.
5. The nucleic acid isolation apparatus according to claim 2, wherein the electro-active hydroxylation treatment is direct current electro-active, the voltage is 1.5 to 1.7V/cm, and the electro-active hydroxylation treatment time is 1 to 2 hours.
6. The nucleic acid extraction apparatus according to claim 1, wherein the silica microspheres are micron-sized silica microspheres.
7. The nucleic acid extraction device according to claim 1, characterized in that the core rod (2) is screwed or slidingly connected to the extraction tube (1).
8. A method for extracting nucleic acid, comprising the steps of:
s1, mixing a nucleic acid sample with a lysis solution to fully lyse the sample;
s2, adding the mixed solution obtained in the step S1 into an extraction tube (1) of the nucleic acid extraction device according to any one of claims 1-7, uniformly mixing the modified silica microspheres, and discharging waste liquid from a liquid outlet (12);
s3, adding a rinsing liquid into the extraction tube (1), uniformly mixing the modified silicon dioxide microspheres, discharging waste liquid from the liquid outlet (12), and washing off impurities attached to the surface of the modified silicon dioxide;
s4, adding an eluent into the extraction tube (1), uniformly mixing the modified silica microspheres, discharging the modified silica microspheres from the liquid outlet (12), and collecting the eluent to obtain purified nucleic acid.
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| CN113278609A (en) * | 2021-06-03 | 2021-08-20 | 广西产研院生物制造技术研究所有限公司 | Regeneration method of silicon substrate nucleic acid purification column |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113278609A (en) * | 2021-06-03 | 2021-08-20 | 广西产研院生物制造技术研究所有限公司 | Regeneration method of silicon substrate nucleic acid purification column |
| CN113278609B (en) * | 2021-06-03 | 2024-02-13 | 广西产研院生物制造技术研究所有限公司 | Regeneration method of silicon substrate nucleic acid purification column |
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