CN103820431A - Nucleic acid extraction and purification method based on nanometer magnetic beads and kit - Google Patents
Nucleic acid extraction and purification method based on nanometer magnetic beads and kit Download PDFInfo
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- CN103820431A CN103820431A CN201410062784.0A CN201410062784A CN103820431A CN 103820431 A CN103820431 A CN 103820431A CN 201410062784 A CN201410062784 A CN 201410062784A CN 103820431 A CN103820431 A CN 103820431A
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Abstract
The invention discloses a nucleic acid extraction and purification method based on nanometer magnetic beads, comprising the following steps: mixing a biological sample and a lysis buffer to make nanometer magnetic beads in the lysis buffer and nucleic acid DNA/RNA which moves into the lysis buffer form a magnetic bead-nucleic acid compound; transferring the compound under the action of a magnetic field to a washing buffer to wash off impurities on the magnetic bead-nucleic acid compound; and transferring the washed magnetic bead- nucleic acid compound under the action of the magnetic field to an elution buffer so as to elute and recover nucleic acid. The nanometer magnetic beads used in the invention have advantages of uniform size, smooth surface, large surface area ratio, high adsorption capacity of nucleic acid, fast magnetic response speed and rapid separation, and can be stored together with the lysis buffer at room temperature for a long time. The extracted nucleic acid DNA/RNA has high purity, is complete and can be directly used for follow-up detection. The method provided by the invention has shorter nucleic acid extraction time than a general magnetic bead method by the use of a nucleic acid extraction reagent, is more suitable for automation and is adopted to realize high-flux nucleic acid DNA/RNA extraction.
Description
Technical field
The present invention relates to paramagnetic particle method and extract purification of nucleic acid field, especially relate to a kind of nucleic acid extraction purification process and test kit based on nanometer magnetic bead.
Background technology
Nucleic acid comprises thymus nucleic acid (RNA) and Yeast Nucleic Acid (DNA) two classes, and oneself knows that nucleic acid is the genetic material of all living things body, is mainly present in nucleus, within virus is present in viral capsid at cell.At present, all be unable to do without extraction and the purifying of nucleic acid in biomedical every field, applying biological nano material, it is the basis of biological subject that quick, high-throughput isolation purifying obtains object nucleic acid DNA/RNA that high purity is complete.
Just because of this, method for extracting nucleic acid is varied, as the method for organic solvent extraction such as phenol/chloroform, resin method, glass powder absorption method, the pellosil adsorption column method of current main-stream all because nucleic acid DNA/RNA concentration purity of injury, complex operation or the extraction of organic solvent to human body low, realize many reasons such as automatization difficulty, unit time flux are low and form the bottleneck into biology high speed development.
Nanometer magnetic bead method, can fast separating and purifying nucleic acid DNA/RNA under the effect of additional magnetic force, because of safety and easily be automated and develop rapidly.Nanometer magnetic bead method for extracting nucleic acid refers to take superparamagnetism monox nanometer magnetic microsphere (hereinafter to be referred as magnetic bead) as carrier, in high salt, low PH solution, adsorb nucleic acid by magnetic bead, and the principle in low salts solution, nucleic acid being departed from from magnetic bead surfaces is carried out the method for nucleic acid extraction, the diameter of normally used nano level magnetic bead is between 100nm～800nm.Due to the following characteristics of magnetic bead, make paramagnetic particle method method for extracting nucleic acid be highly suitable for automation application.
Nanometer magnetic bead is the spherical particle with certain magnetic and special surface structure being formed by Material claddings such as the magnetic microsphere such as Z 250 or ferric oxide and the various silicon-dioxide containing activity functional groups.By polymerization to magnetic microsphere surface increase difference in functionality group as-COOH ,-OH etc., also can covalent attachment enzyme, the biologically active substance such as cell, antibody, just magnetic bead has been endowed various active function, form multiple character magnetic bead.Contrast plain particles material, spherical magnetic bead has good surface volume effect ratio, and selective adsorption capacity is large, and time of equilibrium adsorption is short.When the particle diameter of Z 250 or ferric oxide crystal is less than certain value, magnetic bead has just had good instantaneous magnetic responsiveness, and the superparamagnetism that namely we often say can avoid the such magnetic of particle in occurring to reunite; Because have superparamagnetism, magnetic bead can be positioned, lead and separate under the effect of externally-applied magnetic field.
Existing magnetic bead extraction method need to depend on Proteinase K, and extraction step is more complicated, so required extraction time will be relatively long, also there is the problem that purity is not too high in the sample extracting in addition, and the sample after therefore purifying can not meet follow-up requirement of experiment completely.
Summary of the invention
The object of the invention is to overcome the defect of prior art, a kind of nucleic acid extraction purification process and test kit based on nanometer magnetic bead is provided, realizing fast, high-throughput, extract purification of nucleic acid DNA/RNA to automatization.
For achieving the above object, the present invention proposes following technical scheme: the nucleic acid extraction purification process based on nanometer magnetic bead, comprises the following steps:
1) in biological sample, add lysis buffer, cells in sample, nucleus are broken, nucleic acid DNA/RNA separates with nucleoprotein, nucleoprotein distortion precipitation, nucleic acid DNA/the RNA in described biological sample is isolated in cracking, and the nanometer magnetic bead of described nucleic acid DNA/RNA in described lysis buffer is combined, under outside the action of a magnetic field, nanometer magnetic bead is assembled, and forms magnetic bead-nucleic acid complexes;
2) in described magnetic bead-nucleic acid complexes, add lavation buffer solution, the impurity on magnetic bead-nucleic acid complexes is removed in washing, and under outside the action of a magnetic field, nanometer magnetic bead is assembled, and collects the magnetic bead-nucleic acid complexes after washing;
3) in the magnetic bead-nucleic acid complexes after described washing, add elution buffer, elute recovery in connection with the nucleic acid DNA/RNA on nanometer magnetic bead, obtain the nucleic acid DNA/RNA of purifying.
Preferably, described lysis buffer comprises: sodium iodide 1.5～3M, Guanidinium hydrochloride 2～3M, EDTA1～10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%, pH value=7.4 of described lysis buffer;
Described lavation buffer solution comprises: EDTA1～10mM, Tris-cl150mM, ethanol 75%, pH value=6.4 of described lavation buffer solution;
Described elution buffer adopts 1mM caustic lye of soda.
Described biological sample includes cell liquid sample and acellular liquid sample, described have cell liquid sample to comprise cell, whole blood, animal tissues's homogenate, and described acellular liquid sample comprises serum, blood plasma, tissue extract, swab washing lotion, urine, virus-culturing fluid.
Described nanometer magnetic bead is superparamagnetism monox nanometer magnetic micro-beads, and diameter is 100～800nm, has nucleocapsid structure, i.e. superparamagnetism core and silicon oxide shell.
The present invention also provides a kind of nucleic acid extraction purification kit based on nanometer magnetic bead, component in described test kit comprises lysis buffer, lavation buffer solution and elution buffer, described lysis buffer comprises: sodium iodide 1.5～3M, Guanidinium hydrochloride 2～3M, EDTA1～10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%, pH value=7.4 of described lysis buffer, it (is mainly Guanidinium hydrochloride that described Virahol has strengthened lysis buffer middle and high concentration salt, sodium iodide) effect of leading nanometer magnetic bead absorption nucleic acid, Guanidinium hydrochloride, sodium iodide not only has the effect that promotes nanometer magnetic bead and nucleic acid combination, the effect of dissociating in addition nucleic acid DNA/RNA and nucleoprotein.
Described lavation buffer solution comprises: EDTA1～10mM, Tris-cl150mM, ethanol 75%; Described elution buffer adopts 1mM caustic lye of soda, pH value=6.4 of described lavation buffer solution, wherein, described ethanol has not only strengthened the effect of lavation buffer solution nanometer magnetic bead absorption nucleic acid under low PH condition, also has the effect of dissociate Guanidinium hydrochloride and residual organic matter matter.
Described elution buffer adopts 1mM caustic lye of soda, does not contain alcohols material in described elution buffer.
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 extraction and only need 5-10 minute, as being used in conjunction with self-reacting device, extract 32 biological samples, comprising application of sample only needs 10 minutes interior.And this kit method has overcome the various shortcoming of existing paramagnetic particle method method for extracting nucleic acid step; Cracking and integrating step one step complete, nanometer magnetic bead is directly placed in lysis buffer, and nanometer magnetic bead is existed in reaction soln in the beginning step of nucleic acid extraction, has reduced operation steps, thereby corresponding operation steps, has improved unit time nucleic acid DNA/RNA and has extracted flux.More existing paramagnetic particle method method for extracting nucleic acid, the method is more suitable for full-automatic nucleic acid extraction application; (2) the method is not used Proteinase K, settling agent in the middle of cracking process, cracking is without heating, reduce the performance requriements to self-reacting device, the very big cost that reduced compared with existing paramagnetic particle method nucleic acid extracting reagent, reduce operation steps, reduce the probability of makeing mistakes, and avoided the existing paramagnetic particle method nucleic acid extracting reagent shortcoming of necessary-20 ℃ of preservations of composition in whole or in part; (3) the method is simple, and test kit cost is low, and it is high that the nucleic acid of extraction completes purity, can directly carry out PCR and the research of RT-PCR equimolecular biological experiment and clinical detection; (4 test kits of the present invention can coordinate self-reacting device to use, and fast high-flux extracts the nucleic acid DNA/RNA in biological sample.
Accompanying drawing explanation
Fig. 1 adopts the whole blood genome extraction method efficiency ratio of nucleic acid extraction method of the present invention and QIAamp DNA Mini Kit compared with schematic diagram;
Fig. 2 adopts the whole blood genome extraction method efficiency ratio of nucleic acid extraction method of the present invention and Qiagen pellosil adsorption column method (QiagenViral RNAMini Kit) compared with schematic diagram;
Fig. 3 adopts the whole blood genome extraction method efficiency ratio of nucleic acid extraction method of the present invention and existing paramagnetic particle method nucleic acid extracting reagent compared with schematic diagram.
Embodiment
Below in conjunction with accompanying drawing of the present invention, the technical scheme of the embodiment of the present invention is carried out to clear, complete description.
Embodiment 1
1) get EDTA anticoagulation 200ul and be placed in centrifuge tube, in centrifuge tube, add lysis buffer 600ul, room temperature mixes 3 minutes, then centrifuge tube is placed on magnetic frame, and magneticseparation 20s inhales and abandons supernatant;
Wherein, the composition adding in described lysis buffer respectively: sodium iodide 2M, Guanidinium hydrochloride 2.5M, EDTA10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%, all the other compositions are water, the pH value of lysis buffer is adjusted to 7.4.
2) continue to the lavation buffer solution that adds 400ul in described centrifuge tube, centrifuge tube is placed on magnetic frame after mixing 1 minute, magneticseparation 20s, inhales and abandons supernatant, cool the putting 1 minute of uncapping;
Wherein, the composition adding in described lavation buffer solution respectively: EDTA5mM, Tris-cl150mM, ethanol 75%, all the other compositions are water, the pH value of lavation buffer solution is adjusted to 6.4.
3) repeat above-mentioned 2) operation steps;
4) in the most backward centrifuge tube, add the elution buffer of 50ul, after mixing 1 minute, centrifuge tube is placed on magnetic frame, magneticseparation 20s, supernatant liquor is transferred in the centrifuge tube of another cleaning without enzyme, at-20 ℃ of temperature, preserves the Whole Blood Genomic DNA solution that can obtain finally extracting purifying.
Described elution buffer adopts 1mM caustic lye of soda, and the consumption of elution buffer need to be optional between 50～100 μ l according to user.
The sample size that contrast agents selects QIAamp DNA Mini Kit to extract is 200ul, and elution volume is 100ul.
After extraction completes, get respectively Whole Blood Genomic DNA solution 5ul and do 1.5% agarose gel electrophoresis, result as shown in Figure 1.
1) get containing the serum 100ul of hepatitis C virus (HCV virus) and be placed in centrifuge tube, in centrifuge tube, add lysis buffer 300ul, room temperature mixes 3 minutes, then centrifuge tube is placed on magnetic frame, and magneticseparation 20s inhales and abandons supernatant;
Wherein, the composition adding in described lysis buffer respectively: sodium iodide 2M, Guanidinium hydrochloride 2.5M, EDTA10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%, all the other compositions are water, the pH value of lysis buffer is adjusted to 7.4.
2) continue to the lavation buffer solution that adds 400ul in described centrifuge tube, centrifuge tube is placed on magnetic frame after mixing 1 minute, magneticseparation 20s, inhales and abandons supernatant, cool the putting 1 minute of uncapping;
Wherein, the composition adding in described lavation buffer solution respectively: EDTA5mM, Tris-cl150mM, ethanol 75%, all the other compositions are water, the pH value of lavation buffer solution is adjusted to 6.4.
3) in the most backward centrifuge tube, add the elution buffer of 100ul, after mixing 1 minute, centrifuge tube is placed on magnetic frame, magneticseparation 20s, supernatant liquor is transferred in the centrifuge tube of another cleaning without enzyme, at-20 ℃ of temperature, preserves the HCV viral nucleic acid RNA solution that can obtain finally extracting purifying.
Described elution buffer adopts 1mM caustic lye of soda, and the consumption of elution buffer need to be optional between 50～100 μ l according to user.
Component in the nucleic acid extraction purification kit of preparing in the present invention comprises described lysis buffer, lavation buffer solution and elution buffer, wherein the volume ratio of the composition in each component and each composition is identical with above-mentioned steps, be the composition that adds in described lysis buffer respectively: sodium iodide 2M, Guanidinium hydrochloride 2.5M, EDTA10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%, pH value is adjusted to 7.4; The composition adding in described lavation buffer solution is respectively: EDTA5mM, and Tris-cl150mM, ethanol 75%, the pH value of lavation buffer solution is adjusted to 6.4; Described elution buffer adopts 1mM caustic lye of soda.
The present invention also can coordinate self-reacting device with test kit, extracts the nucleic acid DNA/RNA in biological sample fast high-flux, as used NP968 instrument for extracting nucleic acid, uses program below:
After extraction completes, get above-mentioned HCV nucleic acid RNA extracting solution 2ul as template, carry out HCV fluorescent quantitative poly chain reaction (PCR) and detect, detected result sees the following form 1 and accompanying drawing 2:
Table one
In embodiments of the invention 2, the nanometer magnetic bead method method for extracting nucleic acid that uses the present invention to set up, by its experimental result and the Comparison of experiment results that adopts Qiagen Viral RNA Mini Kit, as shown in Figure 2, result shows, the viral RNA extraction efficiency of paramagnetic particle method viral RNA extraction efficiency of the present invention and Qiagen pellosil adsorption column method is in same level, also slightly be better than contrast agents for enriched sample extraction efficiency, therefore, the present invention has very large advantage in full-automatic nucleic acid extraction application aspect.
Embodiment 3
1) get containing the serum 100ul of hepatitis B virus (HBV virus) and be placed in centrifuge tube, in centrifuge tube, add lysis buffer 300ul, room temperature mixes 3 minutes, then centrifuge tube is placed on magnetic frame, and magneticseparation 20s inhales and abandons supernatant;
2) continue to the lavation buffer solution that adds 400ul in described centrifuge tube, centrifuge tube is placed on magnetic frame after mixing 1 minute, magneticseparation 20s, inhales and abandons supernatant, cool the putting 1 minute of uncapping;
3) in the most backward centrifuge tube, add the elution buffer of 100ul, after mixing 1 minute, centrifuge tube is placed on magnetic frame, magneticseparation 20s, supernatant liquor is transferred in the centrifuge tube of another cleaning without enzyme, at-20 ℃ of temperature, preserves the HBV viral nucleic acid RNA solution that can obtain finally extracting purifying.
Wherein, described lysis buffer, lavation buffer solution and the elution buffer using in the embodiment of the present invention 3 is identical with use in embodiment 2, and the test kit adopting is identical with the test kit in embodiment 2.
Identical with embodiment 2 in addition, the embodiment of the present invention 3 also coordinates self-reacting device with test kit, extracts the nucleic acid DNA/RNA of HBV serum virus fast high-flux, as used NP968 instrument for extracting nucleic acid, uses program below:
After extraction completes, get above-mentioned HBV nucleic acid DNA extracting solution 2ul as template, carry out HBV fluorescent quantitative poly chain reaction (PCR) and detect, detected result sees the following form 2 and accompanying drawing 3:
Table 2
In embodiments of the invention 3, the paramagnetic particle method method for extracting nucleic acid that uses the present invention to set up, the experimental result of its experimental result and the extraction of existing use paramagnetic particle method nucleic acid extracting reagent is compared, result shows, existing contrast agents paramagnetic particle method nucleic acid extracting reagent extraction time is 30 minutes, 3 extraction times of the embodiment of the present invention are only 9 minutes, and the extraction efficiency of paramagnetic particle method nucleic acid extraction efficiency of the present invention and existing paramagnetic particle method nucleic acid extracting reagent is in same level, for also slightly excellent contrast agents of enriched sample extraction efficiency, therefore, the present invention has clear superiority in quick nucleic acid extraction application aspect, coordinating automatic nucleic acid extraction apparatus can realize high-throughput nucleic acid extracts.
Technology contents of the present invention and technical characterictic have disclosed as above; but those of ordinary skill in the art still may be based on teaching of the present invention and announcements and are done all replacement and modifications that does not deviate from spirit of the present invention; therefore; protection domain of the present invention should be not limited to the content that embodiment discloses; and should comprise various do not deviate from replacement of the present invention and modifications, and contained by present patent application claim.
Claims (9)
1. the nucleic acid extraction purification process based on nanometer magnetic bead, is characterized in that, comprises the following steps:
1) in biological sample, add lysis buffer, the nucleic acid DNA/RNA in described biological sample is isolated in cracking, and the nanometer magnetic bead of described nucleic acid DNA/RNA in described lysis buffer is combined, and under outside the action of a magnetic field, forms magnetic bead-nucleic acid complexes;
2) in described magnetic bead-nucleic acid complexes, add lavation buffer solution, the impurity on magnetic bead-nucleic acid complexes is removed in washing, under outside the action of a magnetic field, collects the magnetic bead-nucleic acid complexes after washing;
3) in the magnetic bead-nucleic acid complexes after described washing, add elution buffer, elute recovery in connection with the nucleic acid DNA/RNA on nanometer magnetic bead, obtain the nucleic acid DNA/RNA of purifying.
2. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 1, it is characterized in that, described lysis buffer comprises: sodium iodide 1.5～3M, Guanidinium hydrochloride 2～3M, EDTA1～10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%, pH value=7.4 of described lysis buffer;
Described lavation buffer solution comprises: EDTA1～10mM, Tris-cl150mM, ethanol 75%, pH value=6.4 of described lavation buffer solution;
Described elution buffer adopts 1mM caustic lye of soda.
3. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 1, is characterized in that, described biological sample includes cell liquid sample and acellular liquid sample.
4. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 3, is characterized in that, described in have cell liquid sample to comprise cell, whole blood, animal tissues's homogenate.
5. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 3, is characterized in that, described acellular liquid sample comprises serum, blood plasma, tissue extract, swab washing lotion, urine, virus-culturing fluid.
6. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 1 and 2, is characterized in that, described nanometer magnetic bead is superparamagnetism monox nanometer magnetic micro-beads, and diameter is 100 √ 800nm.
7. the nucleic acid extraction purification kit based on nanometer magnetic bead, it is characterized in that, component in described test kit comprises lysis buffer, lavation buffer solution and elution buffer, described lysis buffer comprises: sodium iodide 1.5～3M, Guanidinium hydrochloride 2～3M, EDTA1～10mM, Tween-203%, nanometer magnetic bead 8%, SDS2%, Virahol 35%; Described lavation buffer solution comprises: EDTA1～10mM, Tris-cl150mM, ethanol 75%; Described elution buffer adopts 1mM caustic lye of soda.
8. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 7, is characterized in that pH value=7.4 of described lysis buffer.
9. the nucleic acid extraction purification process based on nanometer magnetic bead according to claim 7, is characterized in that pH value=6.4 of described lavation buffer solution.
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