CN106591297A - Magnetic bead nucleic acid extraction method - Google Patents
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- CN106591297A CN106591297A CN201710113885.XA CN201710113885A CN106591297A CN 106591297 A CN106591297 A CN 106591297A CN 201710113885 A CN201710113885 A CN 201710113885A CN 106591297 A CN106591297 A CN 106591297A
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- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a magnetic bead nucleic acid extraction method. The method includes: adding a cracking buffer solution into a biological sample, and conducting cracking separation of nucleic acid in the biological sample; combining nucleic acid with nano magnetic beads in the cracking buffer solution to form a magnetic bead-nucleic acid compound under the action of an external magnetic field; adding a washing buffer solution into the magnetic bead-nucleic acid compound to conduct centrifugal washing so as to remove impurities from the magnetic bead-nucleic acid compound, and collecting the washed magnetic bead-nucleic acid compound; adding an elution buffer solution into the washed magnetic bead-nucleic acid compound, and adding a disinhibition agent; directly subjecting a mixture of the magnetic bead-nucleic acid compound and the elution buffer solution to subsequent fluorescence quantitative PCR reaction. The method provided by the invention replaces a magnetic frame with centrifugation for adsorption of magnetic beads, and separates eluent and magnetic beads, can set different rotation speeds and time according to different types of nucleic acid so as to thoroughly separate the eluent and magnetic beads, thus improving the extraction concentration and purity.
Description
Technical field
The invention belongs to nucleic acid extraction technical field, more particularly to paramagnetic particle method method for extracting nucleic acid.
Background technology
Paramagnetic particle method nucleic acid extraction is the surface of superparamagnetic nano particle to be improved with nanometer technology and surface is repaiied
After decorations, superparamagnetism silica nanometer magnetic bead is prepared into.The magnetic bead can specifically be recognized on micro interface with nucleic acid molecules
With efficiently combination.Using the superparamagnetism of silica Nano microsphere, in Chaotropic salt (guanidine hydrochloride, guanidinium isothiocyanate etc.) and
In the presence of externally-applied magnetic field, can isolate from the nucleic acid in blood, animal tissue, food, pathogenic microorganism equal samples and RNA
Come, can be applicable to clinical disease diagnosis, transfusion safety, Forensic Identification, environmental microorganism detection, food safety detection, molecule
The multiple fields such as biological study.
Paramagnetic particle method nucleic acid extraction can be generally divided into four steps：Cracking-combine-washing-wash-out.With traditional nucleic acid extraction
Method is compared, and paramagnetic particle method nucleic acid extraction has the advantage that：1), can realize that automation, high-volume are operated, 96 holes are had at present
Nucleic acid automatic extracting instrument, be capable of achieving the process to 96 samples with extraction time of a sample, this feature is substantially excellent
In other extracting methods.2), the simple to operate, used time is short, and whole flow process of extracting only needs four steps, mostly can be in 36-40 minutes
Inside complete, whole flow process of extracting only has four steps, can complete within 36-40 minutes mostly.3), safety non-toxic, does not use tradition
The toxic reagents such as benzene, chloroform in method, the injury to experiment operator is substantially reduced.4), the specificity of magnetic bead and nucleic acid
All get a promotion with reference to the purity and concentration of the nucleic acid for causing to extract.
In prior art, magnetic bead method for extracting nucleic acid will be through multiple wash-out and washing, nucleic acid in extraction process
DNA/RNA easily loses in extraction process, affects the final purity and concentration for extracting nucleic acid DNA/RNA.To magnetic bead-nucleic acid
Add in compound after lavation buffer solution, when washing removes the impurity on magnetic bead-nucleic acid complexes, to be applied to external magnetic field
Effect, easily causes cleaning solution and magnetic bead-nucleic acid complexes are separated not exclusively, and DNA purity and concentration are relatively low, the final sample for extracting
The requirement of test can not be fully met.
The content of the invention
It is an object of the invention to overcome the defect of prior art, there is provided a kind of paramagnetic particle method method for extracting nucleic acid, Ke Yiyou
Effect improves the final DNA purity extracted and concentration, can meet the demand of different tests.
The technical scheme that the present invention is provided is as follows：
The present invention provides a kind of paramagnetic particle method method for extracting nucleic acid, comprises the following steps：S1, the addition cracking in biological sample
Nucleic acid DNA/the RNA in the biological sample is isolated in buffer solution, cracking；Nucleic acid DNA/the RNA and the lysis buffer
In nanometer magnetic bead combine, under outside magnetic fields, formed magnetic bead-nucleic acid complexes；It is S2, compound to the magnetic bead-nucleic acid
Lavation buffer solution is added in thing, centrifuge washing will be carried out in the magnetic bead-nucleic acid complexes under preset rotation speed and Preset Time,
The impurity on magnetic bead-nucleic acid complexes is removed, the magnetic bead-nucleic acid complexes after washing are collected；Repeated centrifugation washed once；S3、
Elution buffer is added in the magnetic bead-nucleic acid complexes after the washing, and adds agent of disinthibiting；Directly by magnetic bead-nucleic acid
The mixture of compound and elution buffer carries out follow-up quantitative fluorescent PCR reaction.
Further, the high salt concentration in the lysis buffer includes following component：2-3M guanidine hydrochlorides, 1-3M sodium iodides and
5-10mM EDTA；The lysis buffer also includes following component：1-2% triton x-100s, 1-2% Tween-20s, 1-2%
SDS, 30-40%% glycogen, 5-10% magnetic beads；PH value=7.4 of the lysis buffer.
Further, the lavation buffer solution includes following component：5-10mM EDTA, 10-50mM Tris-HCl, 75% second
Alcohol；PH value=6.4 of the lavation buffer solution.
Further, the elution buffer adopts 1 × TE.
Further, the nanometer magnetic bead is superparamagnetism silica nano-magnetic microballon, a diameter of 20- of the nanometer magnetic bead
100nM。
Further, the agent of disinthibiting is including APEO Brij58, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate
One or more combination.
Further, the biological sample includes cellular liquid sample and cell free fluid sample.
Further, it is described to have cellular liquid sample to include cell, whole blood, animal tissue's homogenate.
Further, the cell free fluid sample includes serum, blood plasma, tissue extract, swab washing lotion, urine, virus
Nutrient solution.
Compared with prior art, the paramagnetic particle method method for extracting nucleic acid that the present invention is provided, with advantages below：
The present invention carries out centrifuge washing using centrifugation to magnetic bead-nucleic acid complexes, can not only remove magnetic bead-nucleic acid and be combined
Impurity in thing, moreover it is possible to by lavation buffer solution and Beads enrichment, according to different types of nucleic acid can arrange different rotating speeds and
Time, lavation buffer solution and magnetic bead is thoroughly separated, improve the concentration and purity extracted.
Traditionally, biological sample is through cracking and combination, cleaning 1, cleaning 2, wash-out, elution buffer wash-out magnetic bead-core
After sour compound, nucleic acid is obtained.However, the step of wash-out magnetic bead is saved in the present invention, adds agent of disinthibiting, directly carried out
Follow-up PCR reactions, are so not only convenient for operating and reducing the loss in DNA elution processes.
Agent of disinthibiting is added in the present invention, prevents elution buffer from immediately eluting nucleic acid from magnetic bead-nucleic acid complexes
Out, so as to avoid in elution process, there is loss in nucleic acid DNA/RNA, and then avoids affecting finally to extract nucleic acid DNA/RNA
Purity and concentration.
Description of the drawings
Below by clearly understandable mode, preferred embodiment is described with reference to the drawings, to a kind of paramagnetic particle method nucleic acid extraction
The above-mentioned characteristic of method, technical characteristic, advantage and its implementation are further described.
Fig. 1 is the fluorescent quantitative PCR result schematic diagram of four groups of tests in a kind of paramagnetic particle method method for extracting nucleic acid of the invention.
Specific embodiment
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, it is described below the present invention's
Specific embodiment.It should be evident that for those of ordinary skill in the art, in the premise for not paying creative work
Under, other embodiments can also be obtained.
Embodiment 1：
A kind of paramagnetic particle method method for extracting nucleic acid, comprises the following steps：S1, the biological sample of 200 μ l salivas is put into into centrifugation
In pipe, the lysis buffer of 1000 μ l, pH value=7.4 is added to centrifuge tube, room temperature is mixed 3 minutes, then centrifuge tube is placed in into magnetic
On power frame, the nucleic acid DNA/RNA in the biological sample, the nucleic acid DNA/RNA and institute are isolated in magnetic separation 20s, cracking
The nanometer magnetic bead for stating a diameter of 20nM in lysis buffer is combined, and nanometer magnetic bead is that superparamagnetism silica nano-magnetic is micro-
Pearl, under outside magnetic fields, forms magnetic bead-nucleic acid complexes；Supernatant is abandoned in suction；
Wherein, lysis buffer；The composition of lysis buffer is：2M guanidine hydrochlorides, 2M sodium iodides and 10mM EDTA；1%
Triton x-100 (Triton X-100), 1% Tween-20 (Tween-20), 1%SDS (lauryl sodium sulfate), 30% is sugared
Original, 5% magnetic bead；Remaining composition is water.
S2,600 μ l, pH value=6.4 lavation buffer solution are added in centrifuge tube again, be placed in centrifuge tube after mixing 1 minute
On magnetic frame, magnetic separation 20s removes the impurity on magnetic bead-nucleic acid complexes；Supernatant is abandoned in suction, collects the magnetic bead-core after washing
Sour compound；Repeat aforesaid operations step once, and uncap and cool put 1 minute；
Wherein, the composition of lavation buffer solution is：10mM EDTA (ethylenediamine tetra-acetic acid), 50mM Tris-HCl (three (hydroxyl first
Base) aminomethane), 75% ethanol；Remaining composition is water.
50 μ l elution buffers, elution buffer are added to adopt 1 × TE, TE elution buffers in S3, most backward centrifuge tube
It is formulated by Tris (trishydroxymethylaminomethane) and EDTA (ethylenediamine tetra-acetic acid), is mainly used in dissolving nucleic acid, can be stable
Stored DNA and RNA；Centrifuge tube is placed on magnetic frame after mixing 1 minute, magnetic separation 20s is transferred to supernatant another clean
In the net centrifuge tube without enzyme, you can obtain the DNA solution of final extraction purification.
Wherein, agent of disinthibiting is APEO Brij58, or glyceraldehyde-3-phosphate, or dihydroxyacetone phosphate, or polyoxy
Vinethene Brij58 and glyceraldehyde-3-phosphate, or APEO Brij58, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
Embodiment 2：
A kind of paramagnetic particle method method for extracting nucleic acid, comprises the following steps：S1, the biological sample of 200 μ l salivas is put into into centrifugation
In pipe, the lysis buffer of 1000 μ l, pH value=7.4 is added to centrifuge tube, room temperature is mixed 3 minutes, then centrifuge tube is placed in into magnetic
On power frame, the nucleic acid DNA/RNA in the biological sample, the nucleic acid DNA/RNA and institute are isolated in magnetic separation 20s, cracking
The nanometer magnetic bead for stating a diameter of 20nM in lysis buffer is combined, and nanometer magnetic bead is that superparamagnetism silica nano-magnetic is micro-
Pearl, under outside magnetic fields, forms magnetic bead-nucleic acid complexes；Supernatant is abandoned in suction；
Wherein, lysis buffer；The composition of lysis buffer is：2M guanidine hydrochlorides, 2M sodium iodides and 10mM EDTA；1%
Triton x-100 (Triton X-100), 1% Tween-20 (Tween-20), 1%SDS (lauryl sodium sulfate), 30% is sugared
Original, 5% magnetic bead；Remaining composition is water.
S2,600 μ l, pH value=6.4 lavation buffer solution are added in centrifuge tube again, after mixing 1 minute, be in preset rotation speed
12000g and Preset Time are under 5min, centrifuge washing will to be carried out in the magnetic bead-nucleic acid complexes, remove magnetic bead-nucleic acid multiple
Impurity on compound, collects the magnetic bead-nucleic acid complexes after washing, and supernatant is abandoned in suction；Repeated centrifugation washed once；And cool putting of uncapping
1 minute；
Wherein, the composition of lavation buffer solution is：10mM EDTA (ethylenediamine tetra-acetic acid), 50mM Tris-HCl (three (hydroxyl first
Base) aminomethane), 75% ethanol；Remaining composition is water.
50 μ l elution buffers, elution buffer are added to adopt 1 × TE, TE elution buffers in S3, most backward centrifuge tube
It is formulated by Tris (trishydroxymethylaminomethane) and EDTA (ethylenediamine tetra-acetic acid), is mainly used in dissolving nucleic acid, can be stable
Stored DNA and RNA；Centrifuge tube is centrifuged into (12000g, 5min) after mixing 1 minute, supernatant is transferred to into another cleaning without enzyme
Centrifuge tube in, you can obtain the DNA solution of final extraction purification.
Wherein, agent of disinthibiting is APEO Brij58, or glyceraldehyde-3-phosphate, or dihydroxyacetone phosphate, or polyoxy
Vinethene Brij58 and glyceraldehyde-3-phosphate, or APEO Brij58, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
Embodiment 3：
A kind of paramagnetic particle method method for extracting nucleic acid, comprises the following steps：S1, the biological sample of 200 μ l salivas is put into into centrifugation
In pipe, the lysis buffer of 1000 μ l, pH value=7.4 is added to centrifuge tube, room temperature is mixed 3 minutes, then centrifuge tube is placed in into magnetic
On power frame, supernatant is abandoned in magnetic separation 20s, suction；Nucleic acid DNA/the RNA in the biological sample, the nucleic acid are isolated in cracking
DNA/RNA is combined with the nanometer magnetic bead of a diameter of 20nM in the lysis buffer, and nanometer magnetic bead is superparamagnetism silica
Nano-magnetic microballon, under outside magnetic fields, forms magnetic bead-nucleic acid complexes；
Wherein, lysis buffer；The composition of lysis buffer is：2M guanidine hydrochlorides, 2M sodium iodides and 10mM EDTA；1%
Triton x-100 (Triton X-100), 1% Tween-20 (Tween-20), 1%SDS (lauryl sodium sulfate), 30% is sugared
Original, 5% magnetic bead；Remaining composition is water.
S2,600 μ l, pH value=6.4 lavation buffer solution are added in centrifuge tube again, be placed in centrifuge tube after mixing 1 minute
On magnetic frame, magnetic separation 20s removes the impurity on magnetic bead-nucleic acid complexes；Supernatant is abandoned in suction, collects the magnetic bead-core after washing
Sour compound, repeated centrifugation washed once；And uncap and cool put 1 minute；
Wherein, the composition of lavation buffer solution is：10mM EDTA (ethylenediamine tetra-acetic acid), 50mM Tris-HCl (three (hydroxyl first
Base) aminomethane), 75% ethanol；Remaining composition is water.
50 μ l elution buffers, elution buffer are added to adopt 1 × TE, TE elution buffers in S3, most backward centrifuge tube
It is formulated by Tris (trishydroxymethylaminomethane) and EDTA (ethylenediamine tetra-acetic acid), is mainly used in dissolving nucleic acid, can be stable
Stored DNA and RNA；And agent of disinthibiting is added, directly the mixture of magnetic bead-nucleic acid complexes, elution buffer is carried out subsequently
Quantitative fluorescent PCR reaction.
Wherein, agent of disinthibiting is APEO Brij58, or glyceraldehyde-3-phosphate, or dihydroxyacetone phosphate, or polyoxy
Vinethene Brij58 and glyceraldehyde-3-phosphate, or APEO Brij58, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
Embodiment 4：
A kind of paramagnetic particle method method for extracting nucleic acid, comprises the following steps：S1, the biological sample of 200 μ l salivas is put into into centrifugation
In pipe, the lysis buffer of 1000 μ l, pH value=7.4 is added to centrifuge tube, room temperature is mixed 3 minutes, then centrifuge tube is placed in into magnetic
On power frame, supernatant is abandoned in magnetic separation 20s, suction；Nucleic acid DNA/the RNA in the biological sample, the nucleic acid are isolated in cracking
DNA/RNA is combined with the nanometer magnetic bead of a diameter of 20nM in the lysis buffer, and nanometer magnetic bead is superparamagnetism silica
Nano-magnetic microballon, under outside magnetic fields, forms magnetic bead-nucleic acid complexes；
Wherein, lysis buffer；The composition of lysis buffer is：2M guanidine hydrochlorides, 2M sodium iodides and 10mM EDTA；1%
Triton x-100 (Triton X-100), 1% Tween-20 (Tween-20), 1%SDS (lauryl sodium sulfate), 30% is sugared
Original, 5% magnetic bead；Remaining composition is water.
S2,600 μ l, pH value=6.4 lavation buffer solution are added in centrifuge tube again, be in preset rotation speed after mixing 1 minute
12000g and Preset Time are under 5min, centrifuge washing will to be carried out in the magnetic bead-nucleic acid complexes, remove magnetic bead-nucleic acid multiple
Impurity on compound；Supernatant is abandoned in suction, collects the magnetic bead-nucleic acid complexes after washing；Repeated centrifugation washed once；And cool putting of uncapping
1 minute；
Wherein, the composition of lavation buffer solution is：10mM EDTA (ethylenediamine tetra-acetic acid), 50mM Tris-HCl (three (hydroxyl first
Base) aminomethane), 75% ethanol；Remaining composition is water.
50 μ l elution buffers, elution buffer are added to adopt 1 × TE, TE elution buffers in S3, most backward centrifuge tube
It is formulated by Tris (trishydroxymethylaminomethane) and EDTA (ethylenediamine tetra-acetic acid), is mainly used in dissolving nucleic acid, can be stable
Stored DNA and RNA；And agent of disinthibiting is added, directly the mixture of magnetic bead-nucleic acid complexes, elution buffer is carried out subsequently
Quantitative fluorescent PCR reaction.
Wherein, agent of disinthibiting is APEO Brij58, or glyceraldehyde-3-phosphate, or dihydroxyacetone phosphate, or polyoxy
Vinethene Brij58 and glyceraldehyde-3-phosphate, or APEO Brij58, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
Embodiment 1 is the DNA solution that extraction purification is obtained using magnetic force decontamination+supernatant transfer；Embodiment 2 is to utilize
Centrifugation decontamination+supernatant transfer obtains the DNA solution of extraction purification；Embodiment 3 is turned using magnetic force decontamination+mixed liquor
Move；Embodiment 4 is using centrifugation decontamination+mixed liquor transfer.The experimental result data of four embodiments, it is as shown in table 1 below.
Table 1：
CT is a kind of measurement unit for determining a certain local organization of human body or organ density size, commonly referred to as Heng Shi units.
It can be seen from table 1, first group to the 4th group of mean CT-number is respectively 28.12,25.51,23.98, and 22.80, it is known that four groups
DNA concentration size for group 4>Group 3>Group 2>Group 1, it can be deduced that the optimization method of this patent is effective.
It should be noted that above-described embodiment can independent assortment as needed.The above is only the preferred of the present invention
Embodiment, it is noted that for those skilled in the art, in the premise without departing from the principle of the invention
Under, some improvements and modifications can also be made, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of paramagnetic particle method method for extracting nucleic acid, it is characterised in that comprise the following steps：
S1, lysis buffer is added in biological sample, the nucleic acid DNA/RNA in the biological sample is isolated in cracking；It is described
Nucleic acid DNA/RNA is combined with the nanometer magnetic bead in the lysis buffer, under outside magnetic fields, forms magnetic bead-nucleic acid multiple
Compound；
S2, in the magnetic bead-nucleic acid complexes lavation buffer solution is added, by the magnetic under preset rotation speed and Preset Time
Centrifuge washing is carried out in pearl-nucleic acid complexes, the impurity on magnetic bead-nucleic acid complexes is removed, the magnetic bead-nucleic acid after washing is collected
Compound；Repeated centrifugation washed once；
S3, elution buffer is added in the magnetic bead-nucleic acid complexes after the washing, and add agent of disinthibiting；Directly by magnetic
The mixture of pearl-nucleic acid complexes and elution buffer carries out follow-up quantitative fluorescent PCR reaction.
2. paramagnetic particle method method for extracting nucleic acid as claimed in claim 1, it is characterised in that：
High salt concentration in the lysis buffer includes following component：2-3M guanidine hydrochlorides, 1-3M sodium iodides and 5-10mM
EDTA；
The lysis buffer also includes following component：1-2% triton x-100s, 1-2% Tween-20s, 1-2%SDS, 30-
40% glycogen, 5-10% magnetic beads；
PH value=7.4 of the lysis buffer.
3. paramagnetic particle method method for extracting nucleic acid as claimed in claim 1, it is characterised in that：
The lavation buffer solution includes following component：5-10mM EDTA, 10-50mM Tris-HCl, 75% ethanol；
PH value=6.4 of the lavation buffer solution.
4. paramagnetic particle method method for extracting nucleic acid as claimed in claim 1, it is characterised in that：
The elution buffer adopts 1 × TE.
5. paramagnetic particle method method for extracting nucleic acid as claimed in claim 1, it is characterised in that：
The nanometer magnetic bead is superparamagnetism silica nano-magnetic microballon, a diameter of 20-100nM of the nanometer magnetic bead.
6. paramagnetic particle method method for extracting nucleic acid as claimed in claim 1, it is characterised in that：
The agent of disinthibiting including APEO Brij58, glyceraldehyde-3-phosphate, one or more group of dihydroxyacetone phosphate
Close.
7. the paramagnetic particle method method for extracting nucleic acid as described in any one in claim 1～6, it is characterised in that：
The biological sample includes cellular liquid sample and cell free fluid sample.
8. paramagnetic particle method method for extracting nucleic acid as claimed in claim 7, it is characterised in that：
It is described to have cellular liquid sample to include cell, whole blood, animal tissue's homogenate.
9. paramagnetic particle method method for extracting nucleic acid as claimed in claim 7, it is characterised in that：
The cell free fluid sample includes serum, blood plasma, tissue extract, swab washing lotion, urine, virus-culturing fluid.
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| CN107523565A (en) * | 2017-10-27 | 2017-12-29 | 河南牧业经济学院 | The simplification extracting method of polyphenolic plant RNA based on nanometer magnetic bead |
| WO2018157844A1 (en) * | 2017-03-03 | 2018-09-07 | 绍兴迅敏康生物科技有限公司 | Method for extracting nucleic acid substance by using magnetic bead, and reagent |
| CN108517290A (en) * | 2018-05-31 | 2018-09-11 | 西安交通大学 | A kind of five chamber nucleic acid-extracting apparatus and method based on immiscible phase interfacial tension |
| CN108866042A (en) * | 2018-07-17 | 2018-11-23 | 厦门生命互联科技有限公司 | A kind of extracting method of micro RNA |
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| CN112159804A (en) * | 2020-09-16 | 2021-01-01 | 湖南省动物疫病预防控制中心 | Tick nucleic acid extraction method |
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| CN112980832A (en) * | 2021-04-23 | 2021-06-18 | 南方科技大学 | Nucleic acid extraction method and kit for nucleic acid extraction |
| CN114231526A (en) * | 2022-02-23 | 2022-03-25 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
| CN114479129A (en) * | 2021-12-30 | 2022-05-13 | 苏州白垩纪生物科技有限公司 | Reagent for rapidly extracting nucleic acid by using paramagnetic particle method and nucleic acid extraction method |
| CN115210374A (en) * | 2020-03-13 | 2022-10-18 | 美国西门子医学诊断股份有限公司 | Improved vortex generator for agitating fluid during sample preparation |
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