CN105112404B - A kind of preparation method of animal tissue's nucleic acid extraction liquid - Google Patents
A kind of preparation method of animal tissue's nucleic acid extraction liquid Download PDFInfo
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Abstract
A kind of preparation method of animal tissue's nucleic acid extraction liquid, this method include the preparation of magnetic microsphere and the configuration of surface active and final extract solution.Wherein magnetic microsphere first passes through Polycarbosilane cladding nano-scale magnetic powder, then is made by heat treatment and magnetic separation means.Extract solution obtained by preparation in accordance with the present invention, animal tissue can quickly be dissociated and efficiently separate purification DNA, animal epidemic detection field can be widely applied to.
Description
Technical field
The present invention relates to a kind of preparation method of animal tissue's nucleic acid extraction liquid.
Background technology
Animal diseases suffer from important influence to the food security of animal husbandry and the mankind, using molecular biology method
Most accurate result can be obtained by carrying out diagnosis to animal tissue.Committed step is to be extracted from animal tissue sample
Bacterium or the DNA of virus.The DNA of the different types of DNA of bacteria of energy acquisition or variety classes virus, and genome are complete
Property, purity and concentration, be that the primary goal that DNA is used for animal pathogen diagnosis is extracted from animal tissue.
Usually there are pollution, and complex steps in existing DNA extraction process, the extraction process time is longer.Such as perchloric acid
The extracting methods such as sodium method, SDS methods and urea method, but these methods need sample size larger, and the organic matter such as phenol is easy
Remained in DNA solution, particularly can disturb amplification procedure in follow-up PCR amplification procedures, influence amplification so that should
The application of method is subject to certain restrictions.
Magnetic microsphere in the presence of additional magnetic force, can fast separating and purifying nucleic acid DNA/RNA, because of safety and be easily achieved
Automate and develop rapidly.Magnetic microsphere method for extracting nucleic acid refers to the magnetic microsphere of superparamagnetism in high salt, low PH solution
Middle absorption nucleic acid, and the method that the principle for departing from nucleic acid from magnetic microsphere surface in low salt solutions carries out nucleic acid extraction.
Existing magnetic microsphere is typically to be answered by the magnetic cores such as ferroso-ferric oxide or di-iron trioxide and the materials such as silica
Close the spherical particle with certain magnetic formed.Chinese invention patent (CN1217352) discloses a kind of coated with silica
The preparation method of the magnetic microsphere of ferroso-ferric oxide, it uses acidization to deposit silica on ferroso-ferric oxide surface.And add
The acidic materials (HCl) entered can react with magnetic core ferroso-ferric oxide to a certain extent, so that can be to the magnetic of magnetic microsphere
Property produce it is uncertain influence, cause unstable product quality.
The content of the invention
The purpose of the present invention is for some and some problem present in above-mentioned background technology, there is provided a kind of animal tissue
The preparation method of nucleic acid extraction liquid.
According to the preparation method of animal tissue's nucleic acid extraction liquid of the present invention, comprise the following steps：
(1) preparation of magnetic microsphere
(1) by Polycarbosilane solid dissolving into organic benzene kind solvent, emulsion is formed；Nano-scale magnetic powder is added to
In organic alcohols solvent containing surfactant, stir to form suspension；Suspension is added in emulsion, stirring is equal
It is even, obtain mixed liquor；Wherein the mass ratio of Polycarbosilane and magnetic powder is 2-5:1；
(2) mixed liquor is added dropwise in the warm water solution containing crosslinking agent by microporous barrier, water temperature is maintained at 60-80 DEG C；
So that Polycarbosilane crosslinking curing is on magnetic powder surface in mixed liquor；Moisture and organic solvent are gone using vacuum distillation means
Remove, obtain the microspheres with solid of Polycarbosilane coated magnetic powder；
(3) microspheres with solid is carried out at 500-1000 DEG C, under nitrogen or inert gas shielding being incubated 1-2 hours, poly- carbon
The microspheres with solid of porous surface is formed after silane cracking；
(4) microspheres with solid after heat treatment is screened by Magneto separate means, it is micro- filters out qualified finished product magnetic
Ball；
(2) activation of magnetic microsphere
By obtained finished product magnetic microsphere, catalyst and amino agents in step (1) with 1:1-20：5-30 mass ratio
Mixing, holding pH value are 10-13, and temperature is stirring reaction 5-10 hours at 50-80 DEG C, cooling, through Magnetic Isolation, washing, is obtained
The magnetic microsphere of affinity groups is carried to surface；Wherein catalyst is selected from sodium hydroxide or potassium hydroxide, and amino agents are selected from ammonia
Base benzenecarboximidamide or aminophenyl boronic acid.
(3) preparation of extract solution
By the magnetic microsphere after being activated in step (2) and isothiocyanates, guanidine hydrochloride, sodium iodide, EDTA, dodecyl
Amino acid ligand is set to extract solution, and the wherein concentration of isothiocyanates is 3.5-5M, and the concentration of guanidine hydrochloride is 1.5-3M, sodium iodide
Concentration is 1.2-2.5M, and EDTA concentration is 15-30mM, and the concentration of dodecylamino acid is 60-100mM, magnetic microsphere
Mass concentration is 10-15%.
In (1) of step (1), one or more of organic benzene kind solvent in toluene, dimethylbenzene and divinylbenzene
Combination.The mass ratio of Polycarbosilane and organic benzene kind solvent is preferably controlled in 1-1.5:1, in the ratio range, the breast of formation
Liquid energy enough keeps moderate viscosity, in order to follow-up mixing.Organic alcohols solvent is selected from ethanol or ethylene glycol；Surfactant
Selected from dioctyl succinate disulfonate acid, ten sodium alkyl sulfates, the alkyl trimethyl ammonium of bromination ten or the alkylammonium of bromination ten.Above-mentioned surface
Activating agent can dissolve each other with Organic Alcohol, so as to which nano-magnetic powder is fully dispersed and suspend.Wherein magnetic powder be iron,
Cobalt, nickel or their alloy, or Fe3O4.The particle diameter of magnetic powder is preferably 1nm-30nm, in the particle size range
Magnetic powder has superparamagnetism, i.e., after externally-applied magnetic field removes, magnetic powder is without remanent magnetism, so as to assemble.Magnetic
The mass ratio of powder and organic alcohols solvent is preferably 1-3:1, the mass ratio of surfactant and organic alcohols solvent is preferably
0.05-0.2:1.Under these conditions of mixture ratios, magnetic powder can not only disperse well, and have relatively high concentration,
So as to the guarantee provided for the magnetic intensity of final products.
In (2) of step (1), crosslinking agent is selected from N, N- methylene-bisacrylamides or the pyridine of ammonia third, both crosslinking agent energy
Enough miscible with water, the mass ratio of crosslinking agent and water is preferably 0.05-0.2:1.In this step, the aperture of microporous barrier is preferably 1-
50 μm, more preferably 10-30 μm.After entering warm water solution by the drop of microporous barrier, the oil-in-water for forming size uniformity is spherical
Structure, and the Polycarbosilane of surface with rounded structures crosslinks rapidly solidification under crosslinking agent and temperature facilitation, so as to
Uniformly envelope magnetic powder.
In (3) of step (1), microspheres with solid is heat-treated, the Polycarbosilane for being coated on magnetic powder surface occurs
Cracking, some small molecule volatile hydrocarbons are gone out, so as to form porous coating.Wherein heating rate is particularly critical, if
Using common heat treatment heating rate (5-30 DEG C/min), the rate of cleavage of Polycarbosilane is too fast, will foam and tie
Structure avalanche.The present inventor has been surprisingly found that the cracking of Polycarbosilane when using 0.1-2 DEG C/min ultralow heating rate
Slowly carried out, so as to form the ultramicropore in predominantly less than 0.01 μm aperture, this in magnetic microsphere application process to adsorbing
Nucleic acid has very important effect.By heat treatment, the porous bag that main component is carborundum is formed after Polycarbosilane cracking
Coating, not only with larger specific surface area, while there is higher structural strength, and be tightly combined with magnetic powder.
In (4) of step (1), using Magneto separate means, obtained magnetic microsphere is screened, removes no magnetic
Or the unqualified magnetic microsphere that magnetic not enough requires, finally give the qualified magnetic microsphere finished product for meeting magnetic requirements.
By the activation process of step (2), magnetic microsphere surface is combined with the active group of carboxyl and hydroxyl, make it
There is specific adsorption ability to nucleic acid.
In step (3), the composition such as isothiocyanates, guanidine hydrochloride, sodium iodide in extract solution can be carried out to animal tissue
Effectively dissociation, dissociates nucleic acid DNA/RNA, nucleoprotein and salinity.The animal tissue includes heart, liver, lungs, spleen
Or kidney, or whole blood, blood plasma, serum or animal secretions.
Extract solution obtained by preparation in accordance with the present invention, quick dissociation can be carried out to animal tissue and is efficiently divided
From purification DNA, animal epidemic (such as bird flu, aftosa, swine fever, blue otopathy) detection field can be widely applied to.
Embodiment
The preparation method of animal tissue's nucleic acid extraction liquid of the present invention is described below by specific embodiment.This area skill
Art personnel should be appreciated that the embodiments described below is only the exemplary illustration to the present invention, any not for being made to it
Limitation.
Embodiment 1
Polycarbosilane solid 1000g is weighed, is pulverized last, is added in the rotary evaporation bottle for filling 1000g toluene,
Rotation dissolving forms emulsion at 40 DEG C of water-bath.300g ethanol is first added in beaker, adds 20g dioctyl succinate acid
Sodium sulfonate, after stirring, 500g nanometer iron powder (particle diameter is below 30nm) is added in beaker, stirred 10 minutes, shape
Into suspension.The suspension of preparation is slowly added into the emulsion of preparation, stirred, obtains mixed liquor.In collection vessel
Middle addition 1000g deionized waters, and 100g N is added, N- methylene-bisacrylamides, it is heated to 80 DEG C.On collection vessel
Side sets microporous barrier, and the aperture of microporous barrier is 10 μm.Mixed liquor is added dropwise in collection vessel by microporous barrier.Steamed using decompression
Evaporate means to remove the moisture in collection vessel and organic solvent, obtain the microspheres with solid of Polycarbosilane coated magnetic powder.Will
Microspheres with solid is put into heat-treatment furnace, is passed through argon gas protection, is raised to 800 DEG C with 0.5 DEG C/min heating rate, is incubated 1 hour
Afterwards, Temperature fall, the microspheres with solid of porous surface is obtained.Suctioned out using magnet and have magnetic magnetic microsphere, obtain 568g conjunctions
The finished product magnetic microsphere of lattice.
By obtained finished product magnetic microsphere and sodium hydroxide and aminobenzene carbonamidine with 1:3:8 mass ratio is configured to water-soluble
Liquid, it is 10.8 to keep pH value, and temperature is stirring reaction 5 hours at 80 DEG C, cooling, through Magnetic Isolation, washing, obtains surface band
There is the magnetic microsphere of affinity groups.
Magnetic microsphere after activation and isothiocyanates, guanidine hydrochloride, sodium iodide, EDTA, dodecylamino acid are configured
Into extract solution, the wherein concentration of isothiocyanates is 4M, and the concentration of guanidine hydrochloride is 1.8M, and the concentration of sodium iodide is 1.2M, EDTA
Concentration be 18mM, the concentration of dodecylamino acid be 60mM, and the mass concentration of magnetic microsphere is 12%.
Embodiment 2
Polycarbosilane solid 1000g is weighed, is pulverized last, is added to the rotary evaporation for filling 800g divinylbenzenes
In bottle, at 60 DEG C of water-bath rotation dissolving form emulsion.300g ethylene glycol is first added in beaker, adds 30g bromination
Ten alkyl trimethyl ammoniums, after stirring, 300g nano-cobalt powder (particle diameter is below 30nm) is added in beaker, stirring 10
Minute, form suspension.The suspension of preparation is slowly added into the emulsion of preparation, stirred, obtains mixed liquor.Receiving
Collect and 1000g deionized waters are added in container, and add 100g N, N- methylene-bisacrylamides, be heated to 60 DEG C.Collecting
Microporous barrier is set above container, and the aperture of microporous barrier is 30 μm.Mixed liquor is added dropwise in collection vessel by microporous barrier.Utilize
Vacuum distillation means remove the moisture in collection vessel and organic solvent, and the solid for obtaining Polycarbosilane coated magnetic powder is micro-
Ball.Microspheres with solid is put into heat-treatment furnace, is passed through argon gas protection, is raised to 1000 DEG C with 0.2 DEG C/min heating rate, insulation
After 1 hour, Temperature fall, the microspheres with solid of porous surface is obtained.Suctioned out using magnet and have magnetic magnetic microsphere, obtained
Finished product magnetic microsphere qualified 324g.
By obtained finished product magnetic microsphere and potassium hydroxide and aminophenyl boronic acid with 1:5:10 mass ratio is configured to water
Solution, it is 12.5 to keep pH value, and temperature is stirring reaction 10 hours at 60 DEG C, cooling, through Magnetic Isolation, washing, obtains surface
Magnetic microsphere with affinity groups.
Magnetic microsphere after activation and isothiocyanates, guanidine hydrochloride, sodium iodide, EDTA, dodecylamino acid are configured
Into extract solution, the wherein concentration of isothiocyanates is 5M, and the concentration of guanidine hydrochloride is 2.5M, and the concentration of sodium iodide is 2.0M, EDTA
Concentration be 30mM, the concentration of dodecylamino acid be 80mM, and the mass concentration of magnetic microsphere is 15%.
Embodiment 3
Polycarbosilane solid 1000g is weighed, is pulverized last, is added to the rotary evaporation bottle for filling 1000g dimethylbenzene
In, at 50 DEG C of water-bath rotation dissolving form emulsion.300g ethanol is first added in beaker, adds 45g ten alkyl sulfides
Sour sodium, after stirring, by 300g nanometer Fe3O4Powder (particle diameter is below 30nm) is added in beaker, is stirred 10 minutes, shape
Into suspension.The suspension of preparation is slowly added into the emulsion of preparation, stirred, obtains mixed liquor.In collection vessel
Middle addition 1000g deionized waters, and the 100g pyridine of ammonia third is added, it is heated to 80 DEG C.Microporous barrier is set above collection vessel, it is micro-
The aperture of pore membrane is 50 μm.Mixed liquor is added dropwise in collection vessel by microporous barrier.It will be collected and held using the means that are evaporated under reduced pressure
Moisture and organic solvent in device remove, and obtain the microspheres with solid of Polycarbosilane coated magnetic powder.Microspheres with solid is put into heat
In treatment furnace, nitrogen protection is passed through, 600 DEG C is raised to 1 DEG C/min heating rate, after being incubated 1 hour, Temperature fall, obtains
The microspheres with solid of porous surface.Suctioned out using magnet and have magnetic magnetic microsphere, obtain the qualified finished product magnetic microspheres of 331g.
By obtained finished product magnetic microsphere, sodium hydroxide and aminobenzene carbonamidine with 1:2.5:6 mass ratio is configured to water-soluble
Liquid, it is 11.2 to keep pH value, and temperature is stirring reaction 10 hours at 80 DEG C, cooling, through Magnetic Isolation, washing, obtains surface band
There is the magnetic microsphere of affinity groups.
Magnetic microsphere after activation and isothiocyanates, guanidine hydrochloride, sodium iodide, EDTA, dodecylamino acid are configured
Into extract solution, the wherein concentration of isothiocyanates is 5M, and the concentration of guanidine hydrochloride is 2.8M, and the concentration of sodium iodide is 2.1M, EDTA
Concentration be 25mM, the concentration of dodecylamino acid be 75mM, and the mass concentration of magnetic microsphere is 12%.
Embodiment 4
Polycarbosilane solid 1000g is weighed, is pulverized last, is added to the rotary evaporation bottle for filling 1000g dimethylbenzene
In, at 50 DEG C of water-bath rotation dissolving form emulsion.300g ethylene glycol is first added in beaker, adds 40g bromination ten
Alkylammonium, after stirring, 500g nano-nickel powder (particle diameter is below 30nm) is added in beaker, stirred 10 minutes, shape
Into suspension.The suspension of preparation is slowly added into the emulsion of preparation, stirred, obtains mixed liquor.In collection vessel
Middle addition 1000g deionized waters, and the 100g pyridine of ammonia third is added, it is heated to 80 DEG C.Microporous barrier is set above collection vessel, it is micro-
The aperture of pore membrane is 40 μm.Mixed liquor is added dropwise in collection vessel by microporous barrier.It will be collected and held using the means that are evaporated under reduced pressure
Moisture and organic solvent in device remove, and obtain the microspheres with solid of Polycarbosilane coated magnetic powder.Microspheres with solid is put into heat
In treatment furnace, argon gas protection is passed through, 600 DEG C is raised to 0.1 DEG C/min heating rate, after being incubated 2 hours, Temperature fall, obtains
To the microspheres with solid of porous surface.Suctioned out using magnet and have magnetic magnetic microsphere, it is micro- to obtain the qualified finished product magnetic of 538g
Ball.
By obtained finished product magnetic microsphere, potassium hydroxide and aminophenyl boronic acid with 1:2:5 mass ratio is configured to water-soluble
Liquid, it is 11.5 to keep pH value, and temperature is stirring reaction 8 hours at 60 DEG C, cooling, through Magnetic Isolation, washing, obtains surface band
There is the magnetic microsphere of affinity groups.
Magnetic microsphere after activation and isothiocyanates, guanidine hydrochloride, sodium iodide, EDTA, dodecylamino acid are configured
Into extract solution, the wherein concentration of isothiocyanates is 4.2M, and the concentration of guanidine hydrochloride is 2.5M, and the concentration of sodium iodide is 1.5M,
EDTA concentration is 15mM, and the concentration of dodecylamino acid is 100mM, and the mass concentration of magnetic microsphere is 10%.
Claims (9)
1. a kind of preparation method of animal tissue's nucleic acid extraction liquid, comprises the following steps：
(1) preparation of magnetic microsphere
(1) by Polycarbosilane solid dissolving into organic benzene kind solvent, emulsion is formed；By nano-scale magnetic powder be added to containing
In the organic alcohols solvent of surfactant, stir to form suspension；Suspension is added in emulsion, stirred,
Obtain mixed liquor；Wherein the mass ratio of Polycarbosilane and magnetic powder is 2-5:1；
(2) mixed liquor is added dropwise in the warm water solution containing crosslinking agent by microporous barrier, water temperature is maintained at 60-80 DEG C；So that
Polycarbosilane crosslinking curing is on magnetic powder surface in mixed liquor；Moisture and organic solvent are removed using the means that are evaporated under reduced pressure,
Obtain the microspheres with solid of Polycarbosilane coated magnetic powder；Wherein crosslinking agent is selected from N,N methylene bis acrylamide or ammonia third
The mass ratio of pyridine, crosslinking agent and water is 0.05-0.2:1；
(3) microspheres with solid is carried out at 500-1000 DEG C, under nitrogen or inert gas shielding being incubated 1-2 hours, wherein heating up
Speed is 0.1-2 DEG C/min, and the microspheres with solid of porous surface is formed after Polycarbosilane cracking；
(4) microspheres with solid after heat treatment is screened by Magneto separate means, filters out qualified finished product magnetic microsphere；
(2) activation of magnetic microsphere
By obtained finished product magnetic microsphere in (one) and catalyst, amino agents with 1:1-20：5-30 mass ratio mixing, keeps
PH value is 10-13, and temperature is stirring reaction 5-10 hours at 50-80 DEG C, cooling, through Magnetic Isolation, washing, obtains surface band
There is the magnetic microsphere of affinity groups；Wherein catalyst is selected from sodium hydroxide or potassium hydroxide, and amino agents are selected from aminobenzene carbonamidine
Or aminophenyl boronic acid；
(3) preparation of extract solution
By the magnetic microsphere after being activated in step (2) and isothiocyanates, guanidine hydrochloride, sodium iodide, EDTA, dodecylamino
Acid is configured to extract solution, and the wherein concentration of isothiocyanates is 3.5-5M, and the concentration of guanidine hydrochloride is 1.5-3M, the concentration of sodium iodide
For 1.2-2.5M, EDTA concentration is 15-30mM, and the concentration of dodecylamino acid is 60-100mM；The quality of magnetic microsphere
Concentration is 10-15%.
In (1) 2. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), have
One or more combination of the machine benzene kind solvent in toluene, dimethylbenzene and divinylbenzene.
In (1) 3. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
Middle organic alcohols solvent is selected from ethanol or ethylene glycol.
In (1) 4. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
Middle surfactant is selected from dioctyl succinate disulfonate acid, ten sodium alkyl sulfates, the alkyl trimethyl ammonium of bromination ten or the alkane of bromination ten
Base ammonium.
In (1) 5. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
The particle diameter of middle nano-scale magnetic powder is 1nm-30nm.
In (1) 6. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
Middle magnetic powder is iron, cobalt, nickel or their alloy.
In (1) 7. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
Middle magnetic powder is Fe3O4。
In (2) 8. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
The aperture of middle microporous barrier is 1-50 μm.
In (2) 9. of the preparation method of animal tissue's nucleic acid extraction liquid according to claim 1, wherein step (1), its
The aperture of middle microporous barrier is 10-30 μm.
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| CN108531563A (en) * | 2018-02-05 | 2018-09-14 | 深圳市尚维高科有限公司 | The purposes and lysate of porous microsphere and the application method of lysate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1260595A2 (en) * | 1995-07-07 | 2002-11-27 | Toyo Boseki Kabushiki Kaisha | Nucleic acid-bondable magnetic carrier and method for isolating nucleic acid using the same |
| CN1469775A (en) * | 2000-10-18 | 2004-01-21 | 东洋钢钣株式会社 | Particulate support for separation/purification or extraction and process for producing the same |
| CN104528725A (en) * | 2015-01-08 | 2015-04-22 | 厦门大学 | Preparation method of magnetic silicon carbide ceramic nano particles |
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2015
- 2015-09-23 CN CN201510610935.6A patent/CN105112404B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1260595A2 (en) * | 1995-07-07 | 2002-11-27 | Toyo Boseki Kabushiki Kaisha | Nucleic acid-bondable magnetic carrier and method for isolating nucleic acid using the same |
| CN1469775A (en) * | 2000-10-18 | 2004-01-21 | 东洋钢钣株式会社 | Particulate support for separation/purification or extraction and process for producing the same |
| CN104528725A (en) * | 2015-01-08 | 2015-04-22 | 厦门大学 | Preparation method of magnetic silicon carbide ceramic nano particles |
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