CN112646802A - Magnetic bead method nucleic acid extracting solution and preparation method thereof - Google Patents
Magnetic bead method nucleic acid extracting solution and preparation method thereof Download PDFInfo
- Publication number
- CN112646802A CN112646802A CN202011571423.0A CN202011571423A CN112646802A CN 112646802 A CN112646802 A CN 112646802A CN 202011571423 A CN202011571423 A CN 202011571423A CN 112646802 A CN112646802 A CN 112646802A
- Authority
- CN
- China
- Prior art keywords
- chitosan
- solution
- parts
- chloride
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000005291 magnetic effect Effects 0.000 title claims abstract description 34
- 239000011324 bead Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 25
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 15
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 229920001661 Chitosan Polymers 0.000 claims abstract description 64
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims abstract description 48
- 239000002245 particle Substances 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 6
- 239000003480 eluent Substances 0.000 claims abstract description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 22
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 21
- 239000001110 calcium chloride Substances 0.000 claims description 21
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 19
- 238000009210 therapy by ultrasound Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 17
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 15
- 239000011259 mixed solution Substances 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 239000008118 PEG 6000 Substances 0.000 claims description 6
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 229940015043 glyoxal Drugs 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000001694 spray drying Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- HHLFWLYXYJOTON-UHFFFAOYSA-N Glyoxylic acid Natural products OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 claims description 3
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 3
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 claims description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000008280 blood Substances 0.000 abstract description 6
- 210000004369 blood Anatomy 0.000 abstract description 6
- 239000011248 coating agent Substances 0.000 abstract description 5
- 238000000576 coating method Methods 0.000 abstract description 5
- 238000000605 extraction Methods 0.000 abstract description 5
- 230000005298 paramagnetic effect Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- 235000012239 silicon dioxide Nutrition 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000011164 primary particle Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Materials Engineering (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention discloses a nucleic acid extracting solution by a paramagnetic particle method and a preparation method thereof. The extracting solution comprises modified chitosan-coated magnetic beads, binding solution, washing solution and eluent. The extracting solution of the invention comprises modified chitosan-coated magnetic beads. The magnetic beads coated with chitosan modified by polyaluminium chloride can be obtained by modifying the chitosan coating the magnetic beads, so that the extraction yield and purity of human whole blood genome DNA can be remarkably improved.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a nucleic acid extracting solution by a magnetic bead method and a preparation method thereof.
Background
In the traditional nucleic acid extraction, the operation steps are multiple, the separation time is long, and the separation efficiency is low.
In the magnetic bead method for extracting nucleic acid, after the magnetic beads adsorb the nucleic acid, the nucleic acid can be quickly separated under the action of a magnetic field, so that the extraction step is greatly accelerated. In the process, the extracting solution, especially the magnetic beads, is a key factor in the process of extracting the nucleic acid.
The research on the selection and modification of the coating material of the magnetic ferroferric oxide is a research hotspot for preparing magnetic beads.
Disclosure of Invention
The invention researches the chitosan of ferroferric oxide coating material and the modification thereof, and provides a nucleic acid extracting solution comprising modified chitosan-coated magnetic beads.
The invention discloses a magnetic bead method nucleic acid extracting solution, which comprises modified chitosan-coated magnetic beads, a binding solution, a washing solution and an eluent; the modified chitosan-coated magnetic beads are modified by polyaluminium chloride.
The extracting solution comprises four independent components, namely magnetic beads, binding solution, washing solution and eluent.
In some embodiments of the invention, the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8-10 parts of chitosan, 8-10 parts of acetic acid, 8-10 parts of polyaluminium chloride, 1-3 parts of calcium chloride and 1000 parts of water 800-.
In some embodiments of the invention, the binding solution is an aqueous solution of sodium chloride and PEG 6000;
preferably, the concentration of sodium chloride in the binding solution is 1.5-2.5M, and the concentration of PEG6000 is 8-12 wt%.
In some embodiments of the invention, the wash solution is an aqueous ethanol solution, preferably an aqueous 60-65 v/v% ethanol solution.
In some embodiments of the invention, the elution solution is TE buffer, wherein the concentration of Tris-HCl is 10-15mM, the concentration of EDTA is 1mM, and the pH is 8.0-8.5.
The second aspect of the present invention discloses the method for preparing the extract liquid of the first aspect, comprising the following steps:
s1, preparing the chitosan modified by polyaluminium chloride;
s2, preparing the magnetic beads coated with the polyaluminium chloride modified chitosan.
In some embodiments of the present invention, the step of S1 includes the following steps:
s11, mixing water and calcium chloride, and stirring;
s12, adding the mixed chitosan and acetic acid, and stirring;
s13, adding polyaluminum chloride, mixing, stirring, and adsorbing at room temperature for 2-3 h;
and S14, spray drying to obtain solid powder.
In some embodiments of the present invention, the step of S2 includes the following steps:
s21, mixing ferroferric oxide particles with water, and performing ultrasonic treatment to obtain a mixed solution A;
s22, mixing the chitosan modified by polyaluminium chloride, glyoxal and acetic acid water solution, and stirring to obtain a mixed solution B;
s23, mixing the mixed solution A and the mixed solution B, and stirring for 6-8h at room temperature;
s24, drying;
wherein, the S21 and S23 are introduced into the system by nitrogen, and the S24 is dried under the nitrogen atmosphere.
In some embodiments of the present invention, in the step S12, in the preparation of the polyaluminum chloride modified chitosan material, the content C of calcium chloride is determined by the following formula:
wherein a is the content of chitosan, b is a constant, and b is more than or equal to 5 and less than or equal to 12.
In some embodiments of the present invention, in the step S21, the homogeneous inspection of the uniformity of the mixture of ferroferric oxide particles and water after the ultrasonic treatment is performed to determine whether the mixture is qualified includes the following steps:
s21, mixing ferroferric oxide particles with water, and carrying out ultrasonic treatment for 3-5 min; repeating the steps once;
s22, respectively using vectors X1 and X2 to represent the uniformity of the mixed liquid after the ultrasonic treatment and the ultrasonic treatment;
s23, calculating the stability of the vector using the following formula:
the vector X1 has a stability of
The vector X2 has a stability of
Wherein,
s24, if
If the uniformity of the mixed liquid is more than 3.02, the uniformity of the mixed liquid is judged to be unqualified, and the ultrasonic treatment is continuously repeated once until the mixed liquid is qualified.
The beneficial technical effects of the invention are as follows:
the extracting solution of the invention comprises modified chitosan-coated magnetic beads. The magnetic beads coated with chitosan modified by polyaluminium chloride can be obtained by modifying the chitosan coating the magnetic beads, so that the extraction yield and purity of human whole blood genome DNA can be remarkably improved.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The chitosan is purchased from Shanghai Debong chemical Co., Ltd, and the content is more than 90%. The quality inspection information is as follows:
quality control project index value
Arsenic (As), 0.00006
Viscosity, mPa s 50800
Ignition residue (as sulfate) 1.5
Loss on drying, 10.0
Degree of deacetylation 80.095.0
Hydrochloric acid insoluble substance, 1.0
Heavy metal (in Pb), 0.0015.
The ferroferric oxide is purchased from Shanghai Pantian powder materials GmbH, and has an average particle size of 80nm, a purity of 99.9%, and a specific surface area of 50m2(ii) a bulk density of 0.67g/cm3。
The silicon dioxide is purchased from Nanjing Tianxing New Material Co., Ltd, the primary particle size is 20nm, and the specific surface area is 280m2G, bulk density of 0.10g/cm3
Example 1
The preparation method of the nucleic acid extracting solution by the magnetic bead method comprises the following steps:
the method comprises the following steps:
s1, preparing the chitosan modified by polyaluminium chloride;
s2, preparing the magnetic beads coated with the polyaluminium chloride modified chitosan.
The step of S1 includes the steps of:
s11, mixing water and calcium chloride, and stirring;
s12, adding the mixed chitosan and acetic acid, and stirring;
s13, adding polyaluminum chloride, mixing, stirring, and adsorbing at room temperature for 2.5 h;
and S14, spray drying to obtain solid powder.
The step of S2 includes the steps of:
s21, mixing ferroferric oxide particles with water, and performing ultrasonic treatment to obtain a mixed solution A;
s22, mixing the chitosan modified by polyaluminium chloride, glyoxal and acetic acid water solution, and stirring to obtain a mixed solution B;
s23, mixing the mixed solution A and the mixed solution B, and stirring for 7 hours at room temperature;
s24, drying;
wherein, the S21 and S23 are introduced into the system by nitrogen, and the S24 is dried under the nitrogen atmosphere.
The material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 8 parts of acetic acid, 8 parts of polyaluminium chloride, 1 part of calcium chloride and 800 parts of water.
Preparing the polyaluminium chloride modified chitosan:
adding water into a beaker, adding calcium chloride, and stirring for dissolving;
adding acetic acid into a test tube, adding chitosan, mixing, and oscillating and mixing;
adding a mixed solution of chitosan and acetic acid into a calcium chloride aqueous solution, and stirring;
adding polyaluminium chloride, mixing, stirring, and adsorbing at room temperature for 2.5 h;
spray drying to obtain solid powder, namely the chitosan modified by polyaluminium chloride.
Preparing a magnetic bead coated with polyaluminium chloride modified chitosan:
adding 1g of ferroferric oxide into 5ml of water, and carrying out ultrasonic treatment at 200W for 20 min;
taking 1g of prepared polyaluminium chloride modified chitosan, adding 99ml of 1% (v/v) acetic acid aqueous solution, adding 5ml of 5% glyoxal aqueous solution, mixing and stirring.
The two obtained liquids were mixed, stirred at room temperature for 7h, and dried at 50 + -2 deg.C for 30 min. And obtaining the magnetic beads coated with the chitosan modified by the polyaluminium chloride.
Example 2
The difference from the example 1 is that the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 9 parts of acetic acid, 8 parts of polyaluminium chloride, 1 part of calcium chloride and 800 parts of water.
Example 3
The difference from the example 1 is that the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 10 parts of acetic acid, 8 parts of polyaluminium chloride, 1 part of calcium chloride and 800 parts of water.
Example 4
The difference from the example 1 is that the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 8 parts of acetic acid, 8 parts of polyaluminium chloride, 0.5 part of calcium chloride and 800 parts of water.
Example 5
The difference from the example 1 is that the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 8 parts of acetic acid, 8 parts of polyaluminium chloride, 3 parts of calcium chloride and 800 parts of water.
Example 6
The difference from example 1 is that in the step of S12, the content C of calcium chloride in the material for preparing the polyaluminium chloride-modified chitosan was determined by the following formula:
wherein a is the content of chitosan, b is a constant, and b is more than or equal to 5 and less than or equal to 12.
Researches show that the content of calcium chloride in the material for preparing the polyaluminium chloride modified chitosan can obviously influence the adsorption performance of the nucleic acid of the obtained magnetic beads, and the content of the calcium chloride has a certain correlation with the content of the chitosan. By the method of this example, the appropriate amount of calcium chloride in the prepared composition can be better determined.
Example 7
The difference from the embodiment 1 is that in the step S21, the uniformity of the mixture of ferroferric oxide particles and water after ultrasonic treatment is inspected in a homogeneous manner to determine whether the mixture is qualified, and the method includes the following steps:
s21, mixing ferroferric oxide particles with water, and carrying out ultrasonic treatment for 3-5 min; repeating the steps once;
s22, respectively using vectors X1 and X2 to represent the uniformity of the mixed liquid after the ultrasonic treatment and the ultrasonic treatment;
s23, calculating the stability of the vector using the following formula:
the vector X1 has a stability of
The vector X2 has a stability of
Wherein,
s24, if
If the uniformity of the mixed liquid is more than 3.02, the uniformity of the mixed liquid is judged to be unqualified, and the ultrasonic treatment is continuously repeated once until the mixed liquid is qualified.
In the process of coating ferroferric oxide, if the ferroferric oxide particles are not dispersed sufficiently, the adsorption effect of the obtained magnetic beads can be influenced. The method of the embodiment can ensure the dispersion effect of the reaction system, reflect the influence of each component and content, and eliminate the interference of dispersion influence.
Comparative example 1
The difference from example 1 is that in the preparation of magnetic beads coated with chitosan modified with polyaluminium chloride, chitosan not modified with polyaluminium chloride is used.
In the preparation, the chitosan which is not modified by the polyaluminium chloride is treated by the same method as the method for modifying the chitosan by the polyaluminium chloride.
Comparative example 2
The difference from the example 1 is that the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 8 parts of acetic acid, 8 parts of polyaluminium chloride, magnesium chloride with the same calcium molar content as 1 part of calcium chloride and 800 parts of water.
In the preparation, the magnesium chloride is treated by the same method as calcium chloride.
Comparative example 3
The difference from the example 1 is that the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8 parts of chitosan, 8 parts of acetic acid, 8 parts of silicon dioxide, 1 part of calcium chloride and 800 parts of water.
In the preparation, the silicon dioxide is treated by adopting a method of treating polyaluminium chloride.
Performance investigation:
the magnetic beads coated with polyaluminium chloride modified chitosan prepared in the examples and the comparative examples were taken and extracted under parallel conditions to separate human whole blood genomic DNA. Cell lysate of anticoagulated peripheral venous blood was used as a sample. The binding solution is an aqueous solution of sodium chloride and PEG 6000; the concentration of sodium chloride in the binding solution is 2.0M, and the concentration of PEG6000 is 10 wt%. The washing solution is 60 v/v% ethanol water solution. The eluent is TE buffer solution, wherein the concentration of Tris hydrochloric acid is 15mM, the concentration of EDTA is 1mM, and the pH value is 8.2. The yield and purity of the whole blood genomic DNA were compared with those of comparative example 1, and the results are shown in Table 1.
TABLE 1 Effect on extraction of human Whole blood genomic DNA
| Nucleic acid yield/% | A260/A 280 | |
| Example 1 | 164e | 1.13c |
| Example 2 | 175e | 1.13c |
| Example 3 | 181f | 1.28d |
| Example 4 | 146d | 1.13c |
| Example 5 | 153d | 1.12c |
| Comparative example 1 | 100a | 1.00b |
| Comparative example 2 | 114b | 1.03b |
| Comparative example 3 | 126c | 0.86a |
In the same column of data, different lower case letters represent significant differences, and P is less than 0.05
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (10)
1. A magnetic bead method nucleic acid extracting solution is characterized by comprising modified chitosan-coated magnetic beads, binding solution, washing solution and eluent; the modified chitosan-coated magnetic beads are modified by polyaluminium chloride.
2. The extracting solution according to claim 1, wherein the material for preparing the polyaluminium chloride modified chitosan comprises the following components in parts by weight:
8-10 parts of chitosan, 8-10 parts of acetic acid, 8-10 parts of polyaluminium chloride, 1-3 parts of calcium chloride and 1000 parts of water 800-.
3. The extract liquid according to claim 1 or 2, wherein the binding liquid is an aqueous solution of sodium chloride and PEG 6000;
preferably, the concentration of sodium chloride in the binding solution is 1.5-2.5M, and the concentration of PEG6000 is 8-12 wt%.
4. The extract according to any one of claims 1 to 3, wherein the washing solution is an aqueous ethanol solution, preferably an aqueous ethanol solution of 60 to 65 v/v%.
5. The extract according to any one of claims 1 to 4, wherein the elution solution is TE buffer, wherein the concentration of Tris-HCl is 10 to 15mM, the concentration of EDTA is 1mM, and the pH is 8.0 to 8.5.
6. The method for producing an extract liquid according to any one of claims 1 to 5, comprising the steps of:
s1, preparing the chitosan modified by polyaluminium chloride;
s2, preparing the magnetic beads coated with the polyaluminium chloride modified chitosan.
7. The method of claim 6, wherein the step of S1 includes the steps of:
s11, mixing water and calcium chloride, and stirring;
s12, adding the mixed chitosan and acetic acid, and stirring;
s13, adding polyaluminum chloride, mixing, stirring, and adsorbing at room temperature for 2-3 h;
and S14, spray drying to obtain solid powder.
8. The method according to claim 6 or 7, wherein the step of S2 includes the steps of:
s21, mixing ferroferric oxide particles with water, and performing ultrasonic treatment to obtain a mixed solution A;
s22, mixing the chitosan modified by polyaluminium chloride, glyoxal and acetic acid water solution, and stirring to obtain a mixed solution B;
s23, mixing the mixed solution A and the mixed solution B, and stirring for 6-8h at room temperature;
s24, drying;
wherein, the S21 and S23 are introduced into the system by nitrogen, and the S24 is dried under the nitrogen atmosphere.
9. The method according to any one of claims 7 to 8, wherein in the step S12, the content C of calcium chloride in the material for preparing the polyaluminium chloride modified chitosan is determined by the following formula:
wherein a is the content of chitosan, b is a constant, and b is more than or equal to 5 and less than or equal to 12.
10. The method according to any one of claims 7 to 9, wherein in the step S21, the homogeneity of the mixture of ferroferric oxide particles and water after ultrasonic treatment is inspected to determine whether the mixture is qualified, comprising the following steps:
s21, mixing ferroferric oxide particles with water, and carrying out ultrasonic treatment for 3-5 min; repeating the steps once;
s22, respectively using vectors X1 and X2 to represent the uniformity of the mixed liquid after the ultrasonic treatment and the ultrasonic treatment;
s23, calculating the stability of the vector using the following formula:
the vector X1 has a stability of
The vector X2 has a stability of
Wherein,
s24, if
If the uniformity of the mixed liquid is more than 3.02, the uniformity of the mixed liquid is judged to be unqualified, and the ultrasonic treatment is continuously repeated once until the mixed liquid is qualified.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011571423.0A CN112646802A (en) | 2020-12-27 | 2020-12-27 | Magnetic bead method nucleic acid extracting solution and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011571423.0A CN112646802A (en) | 2020-12-27 | 2020-12-27 | Magnetic bead method nucleic acid extracting solution and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112646802A true CN112646802A (en) | 2021-04-13 |
Family
ID=75363112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011571423.0A Pending CN112646802A (en) | 2020-12-27 | 2020-12-27 | Magnetic bead method nucleic acid extracting solution and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112646802A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113293159A (en) * | 2021-06-21 | 2021-08-24 | 清华大学深圳国际研究生院 | Nucleic acid extraction kit and nucleic acid extraction method |
| CN113484516A (en) * | 2021-07-01 | 2021-10-08 | 山东和生菌业科技有限公司 | Quantitative detection method for morchella mycelium |
| CN114653346A (en) * | 2022-04-25 | 2022-06-24 | 长春晨裕生物医疗科技有限公司 | Improved polyaluminium chloride modified chitosan coated magnetic bead and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090215124A1 (en) * | 2005-02-15 | 2009-08-27 | Weidong Cao | Nucleic acid isolation methods and materials and devices thereof |
| CN104450684A (en) * | 2014-12-26 | 2015-03-25 | 南京中科神光科技有限公司 | Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method |
| CN105148869A (en) * | 2015-10-15 | 2015-12-16 | 东华大学 | Preparation method and application of chitosan-calcium adsorption particles |
| CN106478769A (en) * | 2016-09-23 | 2017-03-08 | 广东东阳光药业有限公司 | A kind of purification process of preliminary treatment Chinese hamster ovary celI culture fluid |
-
2020
- 2020-12-27 CN CN202011571423.0A patent/CN112646802A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090215124A1 (en) * | 2005-02-15 | 2009-08-27 | Weidong Cao | Nucleic acid isolation methods and materials and devices thereof |
| CN104450684A (en) * | 2014-12-26 | 2015-03-25 | 南京中科神光科技有限公司 | Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method |
| CN105148869A (en) * | 2015-10-15 | 2015-12-16 | 东华大学 | Preparation method and application of chitosan-calcium adsorption particles |
| CN106478769A (en) * | 2016-09-23 | 2017-03-08 | 广东东阳光药业有限公司 | A kind of purification process of preliminary treatment Chinese hamster ovary celI culture fluid |
Non-Patent Citations (4)
| Title |
|---|
| CHANG, YC等: "Magnetic chitosan nanoparticles: Studies on chitosan binding and adsorption of Co(II) ions", 《REACTIVE & FUNCTIONAL POLYMERS》 * |
| 张敏: "四种核酸提取磁性纳米颗粒的研制及其应用", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 张文艺等: "聚合氯化铝-壳聚糖复合絮凝剂的合成及在蓝藻沼液预处理中的应用", 《环境化学》 * |
| 殷思贝等: "新型壳聚糖纳米氧化铁磁珠的制备和理化性能测定", 《细胞与分子免疫学杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113293159A (en) * | 2021-06-21 | 2021-08-24 | 清华大学深圳国际研究生院 | Nucleic acid extraction kit and nucleic acid extraction method |
| CN113484516A (en) * | 2021-07-01 | 2021-10-08 | 山东和生菌业科技有限公司 | Quantitative detection method for morchella mycelium |
| CN114653346A (en) * | 2022-04-25 | 2022-06-24 | 长春晨裕生物医疗科技有限公司 | Improved polyaluminium chloride modified chitosan coated magnetic bead and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112646802A (en) | Magnetic bead method nucleic acid extracting solution and preparation method thereof | |
| Zhao et al. | A poly (ethylene glycol)-brush decorated magnetic polymer for highly specific enrichment of phosphopeptides | |
| Li et al. | Facile synthesis of boronate-decorated polyethyleneimine-grafted hybrid magnetic nanoparticles for the highly selective enrichment of modified nucleosides and ribosylated metabolites | |
| CN107937391B (en) | Lysate for extracting abscisic acid from human excrement and preparation method and application thereof | |
| CN110204735B (en) | Preparation method and application of magnetic core-hollow porous molecularly imprinted polymer satellite assembly of macrolide antibiotics | |
| EP2778226B1 (en) | CDNA synthesis method | |
| CN105254707B (en) | Polymer material based on dipeptides and its sugar from glycopeptide be enriched in application | |
| Shen et al. | Ti4+-phosphate functionalized cellulose for phosphopeptides enrichment and its application in rice phosphoproteome analysis | |
| US20220396827A1 (en) | Solid phase extraction material and its use for nucleic acid enrichment and detection | |
| CN112710755A (en) | Novel method for analyzing serum/plasma proteome | |
| Bi et al. | Facile synthesis and application of poly (ionic liquid)-bonded silica hybrid materials | |
| Kapustin et al. | Novel composite matrices modified with nanolayers of polymers as perspective materials for separation of biomolecules and bioanalysis | |
| CN114011376B (en) | Metal oxidation affinity chromatography magnetic mesoporous nano material, preparation method and application | |
| CN115722206B (en) | Composite adsorbent for adsorbing lead, preparation method and adsorption method | |
| CN118028599A (en) | Metal recovery agent, metal compound recovery agent, and method for recovering metal or metal compound | |
| CN113351190B (en) | Immobilized metal ion affinity chromatography microsphere material and preparation and application thereof | |
| CN110394164B (en) | Heavy metal ion magnetic imprinted polymer and preparation method thereof | |
| CN114231526B (en) | Method for extracting genome DNA of high-abundance fecal microorganisms | |
| CN110885394A (en) | Triazine group modified macroporous resin and preparation method thereof | |
| CN110655478A (en) | MUBA monomer, magnetic nano composite microsphere, preparation method and application in enrichment of phosphorylated peptide | |
| CN110511314B (en) | Metal chelating polystyrene nanosphere and preparation method and application thereof | |
| CN114653346A (en) | Improved polyaluminium chloride modified chitosan coated magnetic bead and preparation method thereof | |
| CN115591531A (en) | Phosphorylated peptide adsorbent containing functional polymer brush as well as preparation and application thereof | |
| CN115047108B (en) | Rapid high-throughput screening method for antibiotic drug residues in mutton | |
| CN115453031B (en) | Preparation method and application of solid phase extraction column filler special for ribavirin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210413 |