CN103820431B - Nucleic acid extraction purification process and kit based on nanometer magnetic bead - Google Patents
Nucleic acid extraction purification process and kit based on nanometer magnetic bead Download PDFInfo
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- CN103820431B CN103820431B CN201410062784.0A CN201410062784A CN103820431B CN 103820431 B CN103820431 B CN 103820431B CN 201410062784 A CN201410062784 A CN 201410062784A CN 103820431 B CN103820431 B CN 103820431B
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Abstract
Present invention is disclosed a kind of nucleic acid extraction purification process based on nanometer magnetic bead, step includes: mix biological sample and lysis buffer, the nanometer magnetic bead in lysis buffer is made to form magnetic bead nucleic acid complexes with the nucleic acid DNA dissociating in lysis buffer/RNA, it is transferred to lavation buffer solution liquid under magnetic fields, the impurity on magnetic bead nucleic acid complexes is removed in washing, transfers to wash-out in elution buffer and reclaim nucleic acid under magnetic fields.The nanometer magnetic bead even particle size that the present invention uses, smooth surface, surface area fraction is big, and magnetic response speed is fast, can be long-term with the common room temperature preservation of lysis buffer, strong to nucleic acid absorption power, separates rapid；The nucleic acid DNA extracting/RNA purity is high, complete, can be directly used for subsequent detection, and nucleic acid extraction time more general paramagnetic particle method nucleic acid extracting reagent requires time for shorter, is more suitable for automation, it is achieved that the extraction of high-throughput nucleic acid DNA/RNA.
Description
Technical field
The present invention relates to paramagnetic particle method extraction purification nucleic acid field, especially relate to a kind of based on nanometer magnetic bead
Nucleic acid extraction purification process and kit.
Background technology
Nucleic acid includes DNA (RNA) and ribonucleic acid (DNA) two class, and oneself knows that nucleic acid is all
The inhereditary material of organism, is primarily present in nucleus at cell, is present in viral capsid in virus
In.At present, it all be unable to do without extraction and the purifying of nucleic acid, application biology in biomedical every field
Nano material, quick, high-throughput isolation purifies and obtains the complete purpose nucleic acid DNA/RNA of high-purity
It is the basis of biological subject.
Just because of this, method for extracting nucleic acid is varied, as the method for organic solvent extraction such as phenol/chloroform,
Chelating resin method, glass dust absorption method, the pellosil adsorption column method of current main-stream is all because of organic solvent
Low to the nucleic acid DNA/RNA concentration purity of the injury of human body, complex operation or extraction, realize automatically
The bottleneck changed many reasons such as difficulty, unit interval flux is low and become biology high speed development.
Nanometer magnetic bead method under the effect of additional magnetic force, can fast separating and purifying nucleic acid DNA/RNA,
Because of safety and easily be automated and develop rapidly.Nanometer magnetic bead method for extracting nucleic acid refers to super suitable
Magnetic silicon oxide nano-magnetic microsphere (hereinafter referred to as magnetic bead) is carrier, by magnetic bead at high salt, low PH
Solution adsorbs nucleic acid, and in low salt solutions, nucleic acid is carried out nucleic acid from the principle that magnetic bead surfaces departs from
The method extracted, the diameter of the nanoscale magnetic bead generally using is between 100nm～800nm.Due to
The following characteristics of magnetic bead so that paramagnetic particle method method for extracting nucleic acid is highly suitable for automation application.
Nanometer magnetic bead be by the magnetic microsphere such as ferroso-ferric oxide or di-iron trioxide with various containing active function
The Material claddings such as the silica of group there is the spherical micro-of certain magnetic and special surface structure
Grain.Increase difference in functionality group such as-COOH ,-OH etc. by polymerization to magnetic microsphere surface,
Also can the bioactivator such as covalent bond enzyme, cell, antibody, magnetic bead just has been assigned various active
Function, i.e. forms multiple character magnetic bead.Contrast plain particles material, spherical magnetic bead has good table
Face bulk effect ratio, selective adsorption capacity is big, and time of equilibrium adsorption is short.Ferroso-ferric oxide or three oxygen
When changing the particle diameter of two iron crystal less than certain value, magnetic bead is just provided with good instantaneous magnetic responsiveness, also
Being exactly the superparamagnetism that we often say, in can avoiding occurring, magnetic as particle is reunited；Because tool
Having superparamagnetism, magnetic bead can be positioned, guide and separate under the effect of externally-applied magnetic field.
Existing magnetic bead extraction method need to rely on Proteinase K, and extraction step is more complicated, so institute
The extraction time needing will be relatively long, and the sample additionally extracting there is also the less high problem of purity,
Therefore the sample after purifying can not fully meet follow-up requirement of experiment.
Content of the invention
It is an object of the invention to overcome the defect of prior art, a kind of core based on nanometer magnetic bead is provided
Acid method for extraction and purification and kit, to realize quick, high flux, automatically extraction purification nucleic acid
DNA/RNA。
For achieving the above object, the present invention proposes following technical scheme: carry based on the nucleic acid of nanometer magnetic bead
Take purification process, comprise the following steps:
1) in biological sample, add lysis buffer, make cells in sample, nuclear collapse, nucleic acid
DNA/RNA separates with nucleoprotein, and nucleoprotein deformation precipitation, cracking is isolated in described biological sample
Nucleic acid DNA/RNA, the nanometer magnetic bead in described nucleic acid DNA/RNA and described lysis buffer
In conjunction with under outside magnetic fields, nanometer magnetic bead is assembled, and forms magnetic bead-nucleic acid complexes；
2) adding lavation buffer solution in described magnetic bead-nucleic acid complexes, it is multiple that magnetic bead-nucleic acid is removed in washing
Impurity on compound, under outside magnetic fields, nanometer magnetic bead is assembled, and collects the magnetic bead-core after washing
Acid compound；
3) magnetic bead-nucleic acid complexes after described washing adds elution buffer, be incorporated into and receive
Nucleic acid DNA/RNA on rice magnetic bead elutes recovery, i.e. obtains the nucleic acid DNA/RNA purifying.
Preferably, described lysis buffer includes: sodium iodide 1.5～3M, guanidine hydrochloride 2～3M, EDTA
1～10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, isopropanol 35%, described splits
Solve pH value=7.4 of buffer solution；
Described lavation buffer solution includes: EDTA1～10mM, Tris-cl150mM, ethanol 75%, institute
State pH value=6.4 of lavation buffer solution；
Described elution buffer uses 1mM caustic lye of soda.
Described biological sample includes cellular liquid sample and cell free fluid sample, and described have cell liquid
Body sample includes cell, whole blood, animal tissue's homogenate, described cell free fluid sample include serum,
Blood plasma, tissue extract, swab washing lotion, urine, virus-culturing fluid.
Described nanometer magnetic bead is superparamagnetism silica nano-magnetic microballon, a diameter of 100～800nm,
There is core shell structure, i.e. superparamagnetism core and outer silica shell.
Present invention also offers a kind of nucleic acid extraction purification kit based on nanometer magnetic bead, described reagent
Component in box includes lysis buffer, lavation buffer solution and elution buffer, described lysis buffer
Including: sodium iodide 1.5～3M, guanidine hydrochloride 2～3M, EDTA1～10mM, Tween-203%, receive
Rice magnetic bead 8%, SDS2%, isopropanol 35%, pH value=7.4 of described lysis buffer, described different
Propyl alcohol enhances the leading nanometer of lysis buffer middle and high concentration salt (mainly guanidine hydrochloride, sodium iodide)
The effect of magnetic bead absorption nucleic acid, guanidine hydrochloride, sodium iodide not only have promotion nanometer magnetic bead and nucleic acid to combine
Effect, also has the effect of dissociation nucleic acid DNA/RNA and nucleoprotein.
Described lavation buffer solution includes: EDTA1～10mM, Tris-cl150mM, ethanol 75%；Institute
State elution buffer use 1mM caustic lye of soda, pH value=6.4 of described lavation buffer solution, wherein,
Described ethanol not only enhances the effect of lavation buffer solution nanometer magnetic bead absorption nucleic acid under low PH condition,
Also there is the effect of dissociation guanidine hydrochloride and residual organic matter matter.
Described elution buffer does not contains alcohols thing in using 1mM caustic lye of soda, described elution buffer
Matter.
The invention has the beneficial effects as follows: whole nucleic acid extraction processes of (1) the method only need 3 steps,
Complete specific nucleic acid extract have only to 5-10 minute, as with self-reacting device with the use of, extract 32
Individual biological sample, comprising sample-adding only needs 10 minutes interior.And this kit method overcomes existing magnetic
The various shortcoming of pearl method method for extracting nucleic acid step；Cracking completes with integrating step one step, nanometer magnetic bead
It is directly placed in lysis buffer, make nanometer magnetic bead i.e. there is reaction in the beginning step of nucleic acid extraction
In solution, decrease operating procedure, thus corresponding operating procedure, improve unit interval nucleic acid
DNA/RNA extracts flux.More existing paramagnetic particle method method for extracting nucleic acid, the method be more suitable for complete from
Dynamicization nucleic acid extraction is applied；(2) the method does not use Proteinase K, settling agent in the middle of cracking process,
Cracking, without heating, reduces the performance requirement to self-reacting device, with existing paramagnetic particle method nucleic acid extraction
Reagent is compared and is greatly reduced cost, decreases operating procedure, reduces probability of makeing mistakes, and avoids
Necessary-20 DEG C of shortcomings preserving of existing paramagnetic particle method nucleic acid extracting reagent composition in whole or in part；(3) should
Method is simple, kit low cost, and it is high that the nucleic acid of extraction completes purity, can directly enter performing PCR and
The research of RT-PCR equimolecular biological experiment and clinical detection；(4 kits of the present invention can coordinate automatically
Changing instrument to use, fast high-flux extracts the nucleic acid DNA/RNA in biological sample.
Brief description
Fig. 1 is the whole blood gene of nucleic acid extraction method and the QIAamp DNA Mini Kit using the present invention
Group extraction method efficiency comparison schematic diagram；
Fig. 2 is the nucleic acid extraction method using the present invention and Qiagen pellosil adsorption column method (Qiagen
Viral RNAMini Kit) whole blood genome extraction method efficiency comparison schematic diagram；
Fig. 3 is the whole blood base of nucleic acid extraction method and the existing paramagnetic particle method nucleic acid extracting reagent using the present invention
Because of group extraction method efficiency comparison schematic diagram.
Detailed description of the invention
Below in conjunction with the accompanying drawing of the present invention, the technical scheme of the embodiment of the present invention is carried out clear, complete
Whole description.
Embodiment 1
1) take EDTA anticoagulation 200ul to be placed in centrifuge tube, in centrifuge tube, add lysis buffer
600ul, room temperature mixes 3 minutes, then is placed in centrifuge tube on magnetic frame, magnetic separation 20s, and suction is abandoned
Supernatant；
Wherein, the interior composition adding of described lysis buffer is respectively: sodium iodide 2M, guanidine hydrochloride 2.5M,
EDTA10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, isopropanol 35%, its
Remaining composition is water, and the pH value of lysis buffer is adjusted to 7.4.
2) continue to add the lavation buffer solution of 400ul in described centrifuge tube, will be from after mixing 1 minute
Heart pipe is placed on magnetic frame, magnetic separation 20s, inhales and abandons supernatant, uncaps and cool puts 1 minute；
Wherein, in described lavation buffer solution add composition respectively: EDTA5mM, Tris-cl
150mM, ethanol 75%, remaining composition is water, and the pH value of lavation buffer solution is adjusted to 6.4.
3) above-mentioned 2 are repeated) operating procedure；
4) the most backward centrifuge tube adds the elution buffer of 50ul, by centrifuge tube after mixing 1 minute
It is placed on magnetic frame, magnetic separation 20s, supernatant is transferred to that another is clean without in the centrifuge tube of enzyme,
The Whole Blood Genomic DNA solution that i.e. can get final extraction purification is preserved at a temperature of-20 DEG C.
Described elution buffer uses 1mM caustic lye of soda, and the consumption of elution buffer is according to user's need
Will between 50～100 μ l optionally.
The sample size that contrast agents selects QIAamp DNA Mini Kit to extract is 200ul, elutes body
Amass as 100ul.
After extraction completes, take Whole Blood Genomic DNA solution 5ul respectively and do 1.5% Ago-Gel electricity
Swimming, result is as shown in Figure 1.
Embodiment 2
1) take the serum 100ul containing HCV (HCV virus) to be placed in centrifuge tube, to from
Adding lysis buffer 300ul in heart pipe, room temperature mixes 3 minutes, then is placed in centrifuge tube on magnetic frame,
Magnetic separation 20s, inhales and abandons supernatant；
Wherein, in described lysis buffer add composition respectively: sodium iodide 2M, guanidine hydrochloride 2.5M,
EDTA10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, isopropanol 35%, its
Remaining composition is water, and the pH value of lysis buffer is adjusted to 7.4.
2) continue to add the lavation buffer solution of 400ul in described centrifuge tube, will be from after mixing 1 minute
Heart pipe is placed on magnetic frame, magnetic separation 20s, inhales and abandons supernatant, uncaps and cool puts 1 minute；
Wherein, in described lavation buffer solution add composition respectively: EDTA5mM, Tris-cl
150mM, ethanol 75%, remaining composition is water, and the pH value of lavation buffer solution is adjusted to 6.4.
3) the most backward centrifuge tube adds the elution buffer of 100ul, by centrifuge tube after mixing 1 minute
It is placed on magnetic frame, magnetic separation 20s, supernatant is transferred to that another is clean without in the centrifuge tube of enzyme,
The HCV viral nucleic acid RNA solution that i.e. can get final extraction purification is preserved at a temperature of-20 DEG C.
Described elution buffer uses 1mM caustic lye of soda, and the consumption of elution buffer is according to user's need
Will between 50～100 μ l optionally.
In the present invention preparation nucleic acid extraction purification kit in component include described lysis buffer,
Lavation buffer solution and elution buffer, wherein the volume ratio of the composition in each component and each composition with above-mentioned
In step identical, in i.e. described lysis buffer add composition respectively: sodium iodide 2M, guanidine hydrochloride
2.5M, EDTA10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, isopropanol 35%,
PH value is adjusted to 7.4；In described lavation buffer solution, the composition of addition is respectively: EDTA5mM, Tris-cl
150mM, ethanol 75%, the pH value of lavation buffer solution is adjusted to 6.4；Described elution buffer uses
1mM caustic lye of soda.
The present invention also can coordinate self-reacting device with kit, extracts biological sample fast high-flux
Nucleic acid DNA/RNA in product, such as used NP968 instrument for extracting nucleic acid, then uses following program:
After extraction completes, take above-mentioned HCV nucleic acid RNA extract 2ul as template, carry out HCV
Fluorescent quantitative poly chain reaction (PCR) detects, and testing result see table 1 and accompanying drawing 2:
Table one
In embodiments of the invention 2, use the nanometer magnetic bead method method for extracting nucleic acid that the present invention sets up,
By its experimental result and the Comparison of experiment results using Qiagen Viral RNA Mini Kit, such as Fig. 2
Shown in, result shows, the paramagnetic particle method viral RNA extraction efficiency of the present invention is inhaled with Qiagen pellosil
The viral RNA extraction efficiency of attached column method is in same level, also omits for enriched sample extraction efficiency
Being better than contrast agents, therefore, the present invention has very big excellent in full-automatic nucleic acid extraction application aspect
Gesture.
Embodiment 3
1) take the serum 100ul containing hepatitis type B virus (HBV virus) to be placed in centrifuge tube, to from
Adding lysis buffer 300ul in heart pipe, room temperature mixes 3 minutes, then is placed in centrifuge tube on magnetic frame,
Magnetic separation 20s, inhales and abandons supernatant；
2) continue to add the lavation buffer solution of 400ul in described centrifuge tube, will be from after mixing 1 minute
Heart pipe is placed on magnetic frame, magnetic separation 20s, inhales and abandons supernatant, uncaps and cool puts 1 minute；
3) the most backward centrifuge tube adds the elution buffer of 100ul, by centrifuge tube after mixing 1 minute
It is placed on magnetic frame, magnetic separation 20s, supernatant is transferred to that another is clean without in the centrifuge tube of enzyme,
The HBV viral nucleic acid RNA solution that i.e. can get final extraction purification is preserved at a temperature of-20 DEG C.
Wherein, described lysis buffer, lavation buffer solution and the wash-out using in the embodiment of the present invention 3
Buffer solution is identical with use in embodiment 2, kit and the kit in embodiment 2 i.e. using
Identical.
Additionally same as in Example 2, the embodiment of the present invention 3 also coordinates self-reacting device with kit,
Extract the nucleic acid DNA/RNA of HBV serum virus, as used NP968 nucleic acid fast high-flux
Extraction apparatus, then use following program:
After extraction completes, take above-mentioned HBV nucleic acid DNA extract 2ul as template, carry out HBV
Fluorescent quantitative poly chain reaction (PCR) detects, and testing result see table 2 and accompanying drawing 3:
Table 2
In embodiments of the invention 3, use the paramagnetic particle method method for extracting nucleic acid that the present invention sets up, will
The experimental result that its experimental result is extracted with existing use paramagnetic particle method nucleic acid extracting reagent compares, knot
Fruit shows, existing contrast agents paramagnetic particle method nucleic acid extracting reagent extraction time is 30 minutes, and the present invention is real
Executing example 3 extraction time is only 9 minutes, and the paramagnetic particle method nucleic acid extraction efficiency of the present invention with existing
The extraction efficiency of paramagnetic particle method nucleic acid extracting reagent is in same level, for enriched sample extraction efficiency
Also slightly excellent contrast agents, therefore, the present invention extracts application aspect at Rapid nucleic acid and has clear superiority,
Coordinate automatic nucleic acid extraction apparatus can realize that high-throughput nucleic acid extracts.
The technology contents of the present invention and technical characteristic have revealed that as above, but are familiar with those skilled in the art
Member is still potentially based on teachings of the present invention and announcement and makees all replacements without departing substantially from spirit of the present invention and repair
Decorations, therefore, scope should be not limited to the content disclosed in embodiment, and should include various
Without departing substantially from replacement and the modification of the present invention, and covered by present patent application claim.
Claims (5)
1. the nucleic acid extraction purification process based on nanometer magnetic bead, it is characterised in that comprise the following steps:
1) adding lysis buffer in biological sample, the DNA/RNA in described biological sample is isolated in cracking,
Nanometer magnetic bead in described lysis buffer for the described DNA/RNA is combined, and under outside magnetic fields, is formed
Magnetic bead-nucleic acid complexes；
2) adding lavation buffer solution in described magnetic bead-nucleic acid complexes, washing is removed on magnetic bead-nucleic acid complexes
Impurity, under outside magnetic fields, collect the magnetic bead-nucleic acid complexes after washing；
3) magnetic bead-nucleic acid complexes after described washing adds elution buffer, be incorporated on nanometer magnetic bead
DNA/RNA elute recovery, i.e. obtain purify DNA/RNA；
Described lysis buffer includes: sodium iodide 1.5～3M, guanidine hydrochloride 2～3M, EDTA 10mM,
Tween-20 3%, nanometer magnetic bead 8%, SDS 2%, isopropanol 35%, the pH of described lysis buffer
Value=7.4；
Described lavation buffer solution includes: EDTA 5mM, Tris-Cl 150mM, ethanol 75%, described washing
PH value=6.4 of buffer solution；
Described elution buffer uses 1mM caustic lye of soda；
Described nanometer magnetic bead is superparamagnetism silica nano-magnetic microballon, a diameter of 100～800nm.
2. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 1, it is characterised in that
Described biological sample includes cellular liquid sample and cell free fluid sample.
3. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 2, it is characterised in that
Described have cellular liquid sample to include whole blood, animal tissue's homogenate.
4. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 2, it is characterised in that
Described cell free fluid sample includes serum, blood plasma, virus-culturing fluid.
5. the nucleic acid extraction purification kit based on nanometer magnetic bead, it is characterised in that in described kit
Component includes lysis buffer, lavation buffer solution and elution buffer, and described lysis buffer includes: sodium iodide
1.5～3M, guanidine hydrochloride 2～3M, EDTA 10mM, Tween-20 3%, nanometer magnetic bead 8%, SDS 2
%, isopropanol 35%；Described lavation buffer solution includes: EDTA 5mM, Tris-Cl 150mM, ethanol 75
%；Described elution buffer uses 1mM caustic lye of soda；
PH value=7.4 of described lysis buffer；
PH value=6.4 of described lavation buffer solution.
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