CN117264944A - Cracking binding solution for magnetic bead method blood DNA rapid extraction, kit comprising same and application thereof - Google Patents
Cracking binding solution for magnetic bead method blood DNA rapid extraction, kit comprising same and application thereof Download PDFInfo
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- CN117264944A CN117264944A CN202311343841.8A CN202311343841A CN117264944A CN 117264944 A CN117264944 A CN 117264944A CN 202311343841 A CN202311343841 A CN 202311343841A CN 117264944 A CN117264944 A CN 117264944A
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- lysis
- polypropylene glycol
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- magnetic
- whole blood
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- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000005336 cracking Methods 0.000 title description 3
- 230000009089 cytolysis Effects 0.000 claims abstract description 70
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 69
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 69
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 68
- 229920001451 polypropylene glycol Polymers 0.000 claims abstract description 49
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 46
- 239000004094 surface-active agent Substances 0.000 claims abstract description 32
- 239000012530 fluid Substances 0.000 claims abstract description 31
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims abstract description 30
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims abstract description 30
- WPMWEFXCIYCJSA-UHFFFAOYSA-N Tetraethylene glycol monododecyl ether Chemical compound CCCCCCCCCCCCOCCOCCOCCOCCO WPMWEFXCIYCJSA-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229960000789 guanidine hydrochloride Drugs 0.000 claims abstract description 25
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
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- 238000012360 testing method Methods 0.000 claims description 3
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- 150000003863 ammonium salts Chemical group 0.000 claims description 2
- DLFDEDJIVYYWTB-UHFFFAOYSA-N dodecyl(dimethyl)azanium;bromide Chemical compound Br.CCCCCCCCCCCCN(C)C DLFDEDJIVYYWTB-UHFFFAOYSA-N 0.000 claims description 2
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
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Classifications
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- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Genetics & Genomics (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a lysis binding solution for quickly extracting blood DNA by a magnetic bead method, a kit comprising the lysis solution and application thereof. In particular, the invention relates to a lysis binding fluid comprising: sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, gemini surfactant, and polypropylene glycol. The invention also relates to a kit comprising a reagent for preparing the lysis binding solution, and a method for extracting nucleic acid from a biological sample by using the lysis binding solution or the kit, and application of the kit. The lysis binding solution or the kit can realize rapid extraction of nucleic acid, save flow time, reduce aerosol pollution, facilitate operation and lower cost, can be directly used for machine operation and obtain good extraction effect, and has application prospect.
Description
Technical Field
The invention relates to the field of nucleic acid extraction reagents, in particular to a lysis binding solution and application thereof in nucleic acid extraction.
Background
Nucleic acid is one of the most basic substances of various life, plays a role in storing and transmitting genetic information, and plays a decisive role in a series of important life phenomena such as growth, heredity, variation and the like.
Genomic DNA extraction is the basis for PCR amplification, library construction and sequencing work, and specimens commonly used in clinical work include blood, organ tissue and shed cells, saliva and buccal swab, with blood samples being most widely used.
Traditional methods of nucleic acid extraction include phenol extraction, alkaline lysis, CTAB extraction, boiling, and the like. Since 1979 Vogelstein and gillespei used glass fiber for recovering DNA fragments in agarose gel for the first time, novel nucleic acid extraction methods based on solid phase carriers have been developed, including adsorption membrane centrifugation, glass bead adsorption and magnetic bead extraction.
Compared with the adsorption membrane centrifugal column extraction method, the magnetic bead extraction method has good surface effect and volume effect, the surface area of the magnetic beads is increased greatly, the functional group density and the selective adsorption capacity of the microspheres are increased greatly, and the adsorption equilibrium time of the magnetic beads is shortened. In addition, the magnetic beads have stable physical and chemical properties, can resist acid-base solution with certain concentration and microbial degradation, have certain biocompatibility and can not cause obvious damage to organisms.
The magnetic bead extraction method for extracting DNA is widely applied clinically at present. However, the magnetic bead method nucleic acid extraction methods of different manufacturers have the problems of great difference in efficiency, complex operation method of DNA extraction reagent, long extraction time, and insufficient concentration and purity of the obtained DNA. In addition, the whole blood sample is very complex in type, a large amount of substances interfering with nucleic acid extraction are contained, the clinical fresh whole blood proportion is very high besides the frozen whole blood sample, and a great number of kits on the market are often reported to have poor extraction effects on the clinical fresh whole blood.
Chinese patent CN114317522a discloses a whole blood DNA extraction kit and a nucleic acid extraction method, comprising a lysate, a first rinse solution, a second rinse solution, an eluent, and further comprising a red blood cell lysate and a proteinase K buffer. The method comprises the steps of firstly using a red blood cell lysate to lyse red blood cells of a blood sample, then centrifuging to leave white blood cell sediment, and then using proteinase K and the lysate to carry out heating lysis. Then combining with absolute ethyl alcohol by using magnetic beads, then washing for three times, volatilizing to remove alcohol, and finally heating and eluting. The method has complicated steps, can not directly extract the whole blood sample, and can not realize a high-flux automatic process.
Chinese patent CN104017800B discloses a whole blood DNA extraction kit and method. The kit comprises erythrocyte lysate, leukocyte washing liquid, digestion liquid, proteinase K, purification liquid, gDNA salting-out liquid, gDNA washing liquid, gDNA eluent and the like. The invention not only contains erythrocyte lysate, but also uses lithium chloride for further purification. The method can obtain high-purity DNA, but has complex operation and time and labor waste.
In summary, although the prior art does not provide a rapid and simple nucleic acid extraction reagent for whole blood samples, the steps of proteinase K, erythrocyte lysis or centrifugation to precipitate leukocytes are not required. Therefore, development of an automated high throughput method for frozen whole blood and clinical fresh whole blood samples that directly lyses whole blood samples without the need for additional addition of binding fluid and magnetic beads after sample lysis is necessary for future gene detection.
Disclosure of Invention
The invention aims to provide a rapid nucleic acid extraction kit which can be directly connected with a machine aiming at a whole blood sample, is simple and rapid to operate and can extract DNA with high concentration and purity.
In order to achieve the above object of the present invention, the present invention provides in a first aspect a lysis-binding fluid comprising: sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, gemini surfactant, and polypropylene glycol.
In a second aspect, the invention provides a nucleic acid extraction kit comprising reagents for use in preparing a lysis binding fluid according to the first aspect of the invention.
In a third aspect, the present invention provides a method of extracting or purifying nucleic acid from a biological sample using a lysis binding fluid according to the first aspect of the invention or a kit according to the second aspect of the invention.
In a fourth aspect, the present invention provides the use of a lysis conjugate as described in the first aspect or a kit as described in the second aspect of the invention in nucleic acid extraction of a biological sample, in particular a whole blood sample, for example a frozen whole blood sample or a fresh whole blood sample (e.g.a clinical fresh whole blood sample).
Compared with the prior art, the invention has the following beneficial technical effects:
(1) Provides a rapid nucleic acid extraction kit for whole blood samples, which can be matched with an automatic nucleic acid extractor to realize rapid high-flux nucleic acid extraction or purification.
(2) According to the technical scheme provided by the invention, the second cleaning solution does not contain ethanol or isopropanol which is easy to volatilize, volatilization and drying of the alcohol are not needed, the time of a process is saved, and the pollution of aerosol is reduced.
(3) The technical scheme provided by the invention does not contain proteinase K, and is more convenient to operate and lower in cost.
(4) The cracking of the technical scheme provided by the invention is combined with one step, and can be directly carried out on-machine operation.
(5) The technical scheme provided by the invention has good extraction effect on frozen whole blood and clinical fresh whole blood.
Drawings
FIG. 1 is a gel electrophoresis diagram of the extracted DNA of the kit of the present invention and Qiagen QIAamp DNA mini Kits of example 2 of the present invention for 6 frozen whole blood and 6 fresh whole blood of a clinical tumor patient.
Detailed Description
The present application is further described below with reference to examples.
In order to more clearly describe embodiments of the present invention or technical solutions in the prior art, in the following description, different "an embodiment" or "an embodiment" does not necessarily refer to the same embodiment. Various embodiments may be substituted or combined, and other implementations may be obtained from these embodiments by those of ordinary skill in the art without undue burden.
Definition of the definition
Before the present teachings are described in detail, it is to be understood that this disclosure is not limited to particular compositions or process steps as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
When ranges of values are provided herein, unless expressly stated otherwise, the ranges are intended to include the starting and ending values, as well as values or ranges of values therebetween. For example, "from 0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; the range between them is 0.2-0.3, 0.3-0.4, 0.2-0.4; increments therebetween such as 0.25, 0.35, 0.220, 0.325, 0.49; the increment range therebetween is, for example, 0.26-0.39, etc.
It should be understood that there is a implied "about" prior to the temperatures, masses, weights, volume ratios, concentrations, times, etc. discussed in this disclosure such that slight and insubstantial deviations are within the scope of the teachings herein. Generally, the term "about" means an insubstantial change in the amount of a component of a composition that does not have any significant effect on the effect or stability of the composition. Moreover, the use of "including," "comprising," and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the present teachings. To the extent that any material incorporated by reference is inconsistent with the teachings of this disclosure, the teachings will control.
Embodiments in the specification that are described as "comprising" various components are also considered to be "consisting of" or "consisting essentially of" the components unless specifically indicated; embodiments described in the specification as "consisting of" various components are also considered to be "comprising" or "consisting essentially of" the components.
"nucleic acid" refers to a polymeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs, wherein the nucleosides are linked together by phosphodiester bonds or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA or chimeric DNA-RNA polymers or oligonucleotides and analogs thereof. The nucleic acid "backbone" may be comprised of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages. The nucleic acid may include modified bases to alter the function or behavior of the nucleic acid, such as adding 3' -terminal dideoxynucleotides to prevent additional nucleotides from being added to the nucleic acid. Synthetic methods for preparing nucleic acids in vitro are well known in the art, although nucleic acids can be purified from natural sources using conventional techniques.
In this application, "extract," "isolate," or "purify" refers to the removal of one or more components of a sample or the separation of other sample components. The sample component comprises the target nucleic acid, which is often in a generally aqueous solution phase, which may also comprise cellular fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "extract", "isolate" or "purify" does not mean any degree of purification. Typically, at least 70% or at least 80% or at least 90% of the target nucleic acids are isolated or purified from the other sample components.
As described above, the present invention provides in a first aspect a lysis-binding fluid comprising: sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, gemini surfactant, and polypropylene glycol.
In some preferred embodiments, the lysis binding fluid consists of sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, a Gemini surfactant, polypropylene glycol, and water.
In some preferred embodiments, the lysis-binding fluid comprises: 50-500mM (e.g., 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, or 450 mM) sodium maleate, 1-8M (e.g., 2, 3, 4, 5, 6, or 7) guanidine hydrochloride, 1-8M (e.g., 2, 3, 4, 5, 6, or 7) sodium perchlorate, 50-500mM (e.g., 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, or 450 mM) ammonium chloride, 0.1-20% (e.g., 0.2, 0.5, 1.0, 2.0, 5.0, 10, or 15%) Brij 30, 0.2-20% (e.g., 0.3, 0.5, 1.0, 2.0, 5.0, 10, or 15%) Gemini surfactant, 5-30% (e.g., 10, 15, 20, or 25%) polypropylene glycol, wherein the Brij 30, bri surfactant and the polypropylene glycol are present in volume percent concentration. Preferably, the lysis buffer has a pH of 5.0-9.0 (e.g., 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5).
In some preferred embodiments, the lysis-binding fluid comprises: 50-500mM sodium maleate, 1-8M guanidine hydrochloride, 1-8M sodium perchlorate, 50-500mM ammonium chloride, 0.1-20% Brij 30, 0.2-20% Gemini surfactant, 5-30% polypropylene glycol and the balance water. Preferably, the pH of the lysis-binding fluid is between 5.0 and 9.0.
In other preferred embodiments, the lysis-binding fluid comprises: 50-200mM sodium maleate, 1-5M guanidine hydrochloride, 2-6M sodium perchlorate, 100-300mM ammonium chloride, 0.5-10% Brij 30, 0.5-10% Gemini surfactant and 10-25% polypropylene glycol. Preferably, the pH of the lysis-binding fluid is in the range of 5.5-8.5.
In other preferred embodiments, the lysis-binding fluid comprises: 50-200mM sodium maleate, 1-5M guanidine hydrochloride, 2-6M sodium perchlorate, 100-300mM ammonium chloride, 0.5-10% Brij 30, 0.5-10% Gemini surfactant, 10-25% polypropylene glycol and the balance water, and the pH of the lysis-binding solution is 5.5-8.5.
In other preferred embodiments, the lysis-binding fluid comprises: 75-175mM sodium maleate, 1.5-4.5M guanidine hydrochloride, 2-5M sodium perchlorate, 150-250mM ammonium chloride, 1-5% Brij 30, 1-8% Gemini surfactant, and 12.5-22.5% polypropylene glycol. Preferably, the pH of the lysis-binding fluid is between 7 and 8.5.
In other preferred embodiments, the lysis-binding fluid comprises: 75-175mM sodium maleate, 1.5-4.5M guanidine hydrochloride, 2-5M sodium perchlorate, 150-250mM ammonium chloride, 1-5% Brij 30, 1-8% Gemini surfactant, 12.5-22.5% polypropylene glycol and the balance water. Preferably, the pH of the lysis-binding fluid is between 7 and 8.5.
In other preferred embodiments, the lysis binding fluid comprises 125mM sodium maleate, 4M guanidine hydrochloride, 5M sodium perchlorate, 200mM ammonium chloride, 2.5% Brij 30, 2.5% Gemini surfactant, and 20% polypropylene glycol. Preferably, the pH of the lysis buffer is 8.5.
In other preferred embodiments, the lysis-binding fluid comprises 125mM sodium maleate, 4M guanidine hydrochloride, 5M sodium perchlorate, 200mM ammonium chloride, 2.5% Brij 30, 2.5% Gemini surfactant, 20% polypropylene glycol, and the balance water. Preferably, the pH of the lysis buffer is 8.5.
In the cleavage binding solution, sodium maleate can provide a buffer environment with stable pH, maintain the charge state of nucleic acid phosphate groups and protect DNA bases.
Guanidine hydrochloride is a chaotropic agent for destroying the three-dimensional structure of proteins, can quickly destroy cell membranes or viruses, release nucleic acids and denature the proteins, so that the nucleic acids can get rid of the entanglement of the proteins, and meanwhile, guanidine hydrochloride is a strong inhibitor of nuclease, so that the nuclease activity can be reduced and the nuclease activity can be inhibited.
Sodium perchlorate is also a protein denaturant, similar to guanidine hydrochloride, and provides a large number of monovalent cations (sodium ions) to establish the phosphate radical of nucleic acid and the salt bridge of the silicon hydroxyl surface of magnetic beads.
Ammonium chloride cooperates with sodium ions to provide favorable conditions for DNA precipitation, and ammonium ions have better penetrability to cells and can be helpful for rapid cell lysis.
Brij 30 (CAS: 9002-92-0) is a relatively mild nonionic surfactant that dissolves lipids and has the effect of promoting hydrogen bonding and cationic bridge formation, often as an additive to stabilize proteins, particularly membrane proteins in their natural conformation. The present invention in the examples attempted to replace with other conventional nonionic surfactants such as triton X-100 or tween 20, impressively Brij 30 had better cell lysis effect and nucleic acid quality and recovery in blood samples than the two surfactants described above, yielding a clear eluate.
Gemini surfactant is also called as Gemini quaternary ammonium salt, and the Gemini quaternary ammonium salt (Gemini quaternary ammonium salt) is formed by connecting two single-chain single-head group common surfactants at an ion head group through chemical bonds, so that the head group separation force in the orderly aggregation process of the surfactants is suppressed, and the surface activity is greatly improved. The surface of the liquid base is easier to adsorb, so that the surface tension of the aqueous solution is reduced more effectively, the cell membrane can be dissolved rapidly, the protein denaturation is accelerated, and the nucleic acid is released.
The Gemini surfactant is selected from one or a combination of more of octadecylamine polyoxyethylene ether biquaternary ammonium salt, cetyl polyoxyethylene ether dimethyl octyl ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride, ethylene bis (cetyl amide propyl dimethyl ammonium bromide), dodecyl polyoxyethylene ether dimethyl octyl ammonium chloride and dodecyl polyoxyethylene ether dimethyl dodecyl ammonium bromide. Preferably, the Gemini surfactant is dodecyl polyoxyethylene ether dimethyl octyl ammonium chloride.
The polypropylene glycol provided by the invention is used as a nucleic acid precipitating agent, can effectively precipitate nucleic acid under the conditions of alkalescence and chaotropic agents, and has better nucleic acid precipitation effect relative to polyethylene glycol. Surprisingly, the inventors have found that the precipitation of nucleic acids in the system is related to the molecular weight of polypropylene glycol. In some preferred embodiments, the polypropylene glycol has a molecular weight of 200-20000. Preferably, the polypropylene glycol has a molecular weight of 500-10000. More preferably, the polypropylene glycol has a molecular weight of 1000 to 8000. Most preferably, the polypropylene glycol molecular weight is 4000.
In a second aspect, the invention provides a nucleic acid extraction kit comprising reagents for use in preparing a lysis binding fluid according to the first aspect of the invention.
In some preferred embodiments, the reagent for a lysis binding fluid consists of: sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, a Gemini surfactant, and polypropylene glycol. In this case, water such as deionized water may be additionally prepared when the lysis binding solution is formulated using the kit. Of course, water, such as deionized water, may also be provided in the kit for use in formulating the lysis conjugate. Thus, in another preferred embodiment, the reagent for a lysis binding fluid consists of the following components: sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, a Gemini surfactant, polypropylene glycol and water.
In the case of nucleic acid extraction using the kit, the extraction may be performed by a magnetic bead extraction method. In this case, one or more selected from the group consisting of a magnetic bead suspension, a washing liquid, and an elution liquid may be additionally formulated for use in combination with the above-described kit for magnetic bead extraction of nucleic acids. Thus, in some preferred embodiments, the rapid nucleic acid extraction kit further comprises one or more selected from the group consisting of reagents for formulating a magnetic bead suspension, reagents for formulating a wash solution, and reagents for formulating an eluate.
The composition of the magnetic bead suspension, the washing liquid and the eluent is not particularly limited as long as it can be used for extraction of a target biological sample in combination with the lysis conjugate of the present invention or the kit. However, in other preferred embodiments, the magnetic bead suspension comprises a dispersion comprising 0.05 to 0.2M (e.g., 0.06, 0.07, 0.08, 0.09, 0.1 or 0.15M) sodium chloride, 0.01 to 0.05 (e.g., 0.02, 0.03 or 0.04%) NaN, and a mass volume percentage of 2 to 20% (e.g., 5, 10 or 15%) superparamagnetic silicon particles having a particle size of 0.1 to 10 μm (e.g., 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0 μm (), and a pH of the magnetic bead suspension of 4.0 to 7.0 (e.g., 4.5, 5.0, 5, 6.0 or 6.5) 3 And the balance of water, wherein the superparamagnetic silica particlesThe amount of particles in the magnetic bead suspension is in mass volume percent, i.e. the mass of superparamagnetic silicon particles is in volume percent of the magnetic bead suspension.
In some more preferred embodiments, the magnetic bead suspension comprises a dispersion comprising 0.05 to 0.2M (e.g., 0.06, 0.07, 0.08, 0.09, 0.1 or 0.15M) sodium chloride, 0.01 to 0.05 (e.g., 0.02, 0.03 or 0.04%) NaN, and a mass volume percent of superparamagnetic silica particles having a particle size of 0.5 to 5 μm (e.g., 0.6, 0.7, 0.8 or 9%), and a pH of 5.0 to 6.0 (e.g., 5.5) 3 And the balance water.
In some further preferred embodiments, the magnetic bead suspension comprises a dispersion comprising 0.05M sodium chloride, 0.05% NaN, and a mass volume percentage of 5% superparamagnetic silica particles having a particle size of 0.5-5 μm, the pH of the magnetic bead suspension being 5.0 3 And the balance water.
The water used for the magnetic bead suspension is not particularly limited in the present invention as long as it does not adversely affect nucleic acid extraction from a biological sample. However, the water is preferably nuclease-free water.
In other preferred embodiments, the cleaning solution may include two types of cleaning solutions, which may be referred to as a first general purpose cleaning solution and a second general purpose cleaning solution to distinguish the two by name.
In some embodiments, the first universal washing solution comprises 5-1000mM (e.g., 10, 20, 50, 100, 200, 500, or 800 mM) Tris-HCl,10-1000mM (e.g., 20, 50, 100, 200, 500, or 800 mM) lithium chloride, 1-20mM (e.g., 2, 5, 10, or 15 mM) EDTA-Na 2 1-5M (e.g., 2, 3 or 4M) sodium perchlorate and 50-80% (e.g., 55, 60, 65, 70 or 75%) absolute ethanol, pH4-8 (e.g., 5, 6 or 7). Preferably, the first universal washing liquid is 10-500mM Tris-HCl,50-500mM lithium chloride, 2-10mM EDTA-Na 2 2-4M sodium perchlorate and 50-60% absolute ethanol, pH4-7. More preferably, the first universal washing solution comprises 100mM Tris-HCl,200mM lithium chloride, 2mM EDTA-Na 2 3M sodium perchlorate and 50% absolute ethanol, pH5.5.
In other preferred embodiments, the second general purpose cleaning solution comprises: 10-200mM (e.g., 20, 30, 50, 80, 100, or 150 mM) Tris-HCl,10-100mM (e.g., 20, 30, 40, 50, 60, 70, 80, or 90 mM) lithium chloride, and 30-70% (e.g., 40, 50, or 60%) polypropylene glycol 400, pH5-8 (e.g., 5.5, 6.0, 6.5, 7.0, or 7.5). Preferably, the second universal washing solution comprises 20-100mM Tris-HCl,50-100mM lithium chloride and 40-60% polypropylene glycol 400, pH7. More preferably, the second universal washing solution comprises 50mM Tris-HCl,100mM lithium chloride and 50% polypropylene glycol 400, pH7.
In other preferred embodiments, the eluate comprises 10mM Tris-HCl and has a pH of 8.5.
In a second aspect, the invention provides a method of extracting or purifying nucleic acid from a biological sample using a lysis binding fluid according to the first aspect of the invention or a kit according to the second aspect of the invention.
In some preferred embodiments, the method comprises the steps of:
(1) Sample preparation: adding the whole blood sample into a centrifuge tube;
(2) Sample lysis: adding the lysis binding solution and the magnetic bead suspension into the centrifuge tube in the step (1), uniformly mixing and incubating for 5-30min (e.g. 10, 15, 20 or 25 min) at a temperature of 40-80 ℃ (e.g. 50, 60 or 70 ℃);
(3) Nucleic acid adsorption: carrying out magnetic separation by using a magnetic frame, and then discarding liquid obtained after the magnetic separation is completed to obtain magnetic beads after adsorption;
(4) First cleaning: adding the first general cleaning solution into the adsorbed magnetic beads, uniformly mixing, performing magnetic separation by using a magnetic frame, and discarding the liquid obtained after the magnetic separation is completed to obtain first cleaning magnetic beads;
(5) And (3) second cleaning: adding the second general cleaning liquid into the first cleaning magnetic beads, uniformly mixing, then performing magnetic separation by using a magnetic frame, and then discarding the liquid obtained after the magnetic separation is completed to obtain second cleaning magnetic beads;
(6) Sample elution: adding an eluent into the second cleaning magnetic beads, mixing uniformly, then, incubating and eluting at 50-70 ℃ (e.g. 60 ℃) for 5-10min (e.g. 6, 7, 8 or 9 min), and collecting DNA solution obtained after eluting.
The magnetic separation is performed as a routine procedure, and it is well within the ability of those skilled in the art to determine whether the magnetic separation is complete, typically, the beads are fully adsorbed or the liquid becomes clear, i.e., the magnetic separation is considered complete.
In other preferred embodiments, in step (1), the whole blood sample may be used in a volume of 200 μl. In this case, the amount of the lysis buffer added to the biological sample, e.g., whole blood sample, may be 1-3 times (e.g., 2-fold or 2-3 times) the amount of the lysis buffer; the volume of the lysis buffer may be 200-600. Mu.L (e.g., 300, 400, or 500. Mu.L); in step (2), 10-30. Mu.L (e.g., 20. Mu.L) of the magnetic bead suspension may be added; in step (4), 200-1000 μl (e.g. 300, 400, 500, 600, 700, 800 or 900 μl) of the first universal cleaning solution may be added; in step (5), 200-1000 μl (e.g. 300, 400, 500, 600, 700, 800 or 900 μl) of the second universal cleaning solution may be added; in step (6), 40-200. Mu.L (e.g., 50, 100 or 150. Mu.L) of the eluent may be added, and the elution incubated at 50-70℃for 3-6min (e.g., 3, 4 or 5 min) (e.g., 60 ℃).
The biological sample is a whole blood sample, for example, the biological sample is a frozen whole blood or a fresh whole blood sample such as a clinical fresh whole blood sample. For example, in the case where the biological sample is an avian blood sample, in the case where the volume of the biological sample is 200 μl, the avian blood sample may be a 10% by volume aqueous solution obtained by supplementing 20 μl of avian blood with 180 μl of deionized water to obtain an avian blood sample ready for nucleic acid extraction.
In other preferred embodiments, in step (1), the whole blood sample is used in a volume of 200 μl. In step (2), adding 1-3 times the volume of the whole blood sample of the lysis buffer (200-600. Mu.L); in step (2), 10-30. Mu.L of the magnetic bead suspension is added; in step (4), 200-1000 μl of the first universal cleaning solution is added; in step (5), 200-1000 μl of the second universal cleaning solution is added; in step (6), 40-200. Mu.L of the eluent is added, and the eluent is incubated and eluted at 50-70 ℃ for 3-6min.
In other preferred embodiments, in step (1), the whole blood sample is used in a volume of 200 μl. In step (2), adding 2-3 times the volume of the whole blood sample of the lysis buffer (400-600. Mu.L); in step (2), 10-30. Mu.L of the magnetic bead suspension is added; in step (4), 500-700 μl of the first universal cleaning solution is added; in step (5), 500-700 μl of the second universal cleaning solution is added; in step (6), 50-100. Mu.L of the eluent is added, and the eluent is incubated at 60 ℃ for 5min with shaking.
In some more specific embodiments, the present invention provides a kit method for rapid extraction of DNA from a whole blood sample, the method comprising the steps of:
(1) Sample preparation: 200 μl of whole blood sample was added to the centrifuge tube;
(2) Sample lysis: adding 400-600 mu L of the lysis binding solution and 10-30 mu L of the magnetic bead suspension into the centrifuge tube in the step (1), uniformly mixing and incubating for 5-30min at the temperature of 40-80 ℃;
(3) Nucleic acid adsorption: carrying out magnetic separation by using a magnetic frame, and then discarding liquid obtained after the magnetic separation is completed to obtain magnetic beads after adsorption;
(4) First cleaning: adding 500-700 mu L of a first general cleaning solution into the magnetic beads obtained in the step (3), shaking and uniformly mixing for 1min, then placing the mixture on a magnetic rack, and discarding all the liquid obtained after the magnetic separation is completed;
(5) And (3) second cleaning: adding 500-700 mu L of a second general-purpose cleaning solution into the magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing the mixture on a magnetic rack, and then discarding all the liquid obtained after the magnetic separation is completed;
(6) Sample elution: adding 50-100 mu L of eluent into the magnetic beads obtained in the step (5), shaking, incubating and eluting at 60 ℃ for 5min, placing the magnetic beads on a magnetic rack, collecting the DNA solution obtained after eluting after the magnetic beads are adsorbed, and storing at-80 ℃.
In a fourth aspect, the present invention provides the use of a lysis conjugate as described in the first aspect or a kit as described in the second aspect of the invention in nucleic acid extraction, in particular genomic DNA extraction, of a biological sample, in particular a whole blood sample, for example a frozen whole blood sample or a fresh whole blood sample (e.g.a clinical fresh whole blood sample).
Examples
For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the invention, and are not limiting of the claims of the invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified; such materials, unless otherwise specified, are commercially available.
Preparation example 1: rapid nucleic acid extraction kit
The preparation example adopts the rapid nucleic acid extraction kit of the invention to extract nucleic acid. The kit comprises: a reagent for a lysis binding solution for preparing a lysis binding solution, a reagent for a magnetic bead suspension for preparing a magnetic bead suspension, a first cleaning reagent for preparing a first general-purpose cleaning solution, a second cleaning reagent for preparing a second general-purpose cleaning solution, and a reagent for an eluent for preparing an eluent.
1) The composition of the lysis binding solution is as follows: the lysis conjugate contained 125mM sodium maleate, 4M guanidine hydrochloride, 5M sodium perchlorate, 200mM ammonium chloride, 2.5% Brij 30, 2.5% Gemini surfactant, 20% polypropylene glycol (molecular weight 4000) and the balance water, and the pH of the lysis conjugate was 8.5.
2) The magnetic bead suspension comprises a dispersion liquid and superparamagnetic silicon particles with the mass volume percentage of 5 percent and the particle diameter of 0.5-5 mu M, the pH value of the magnetic bead suspension is 5.0, and the dispersion liquid comprises 0.05M sodium chloride and 0.05 percent NaN 3 And the balance water. The superparamagnetic Silica particles were from MagH1N Silica (cat#bmd 00751) of the soviet chalk biotech limited.
3) The first general cleaning liquid consists of the following components: 100mM Tris-likeHCl,200mM lithium chloride, 2mM EDTA-Na 2 3M sodium perchlorate and 50% absolute ethanol, pH5.5.
4) The second general cleaning liquid consists of the following components: 50mM Tris-HCl,100mM lithium chloride and 50% polypropylene glycol 400.
5) The eluent comprises the following components: an aqueous solution (pH 8.5) containing 10mM Tris-HCl.
Comparative preparation 1:
the cleavage bond solution contained no polypropylene glycol and the remaining components were identical to those of preparation example 1.
Comparative preparation 2:
brij 30 in the lysate was replaced with an equal volume of tween 20 and the remaining components were identical to those of preparation 1.
Comparative preparation 3:
brij 30 in the lysis conjugate was replaced with an equal volume of triton X-100, and the remaining components were identical to those of preparation 1.
Comparative preparation 4:
the polypropylene glycol molecular weight in the cleavage bonding liquid was 1000, and the remaining components were the same as those in preparation example 1.
Comparative preparation 5:
the polypropylene glycol molecular weight in the cleavage bonding solution was 2000, and the remaining components were the same as those in preparation example 1.
Comparative preparation 6:
the polypropylene glycol molecular weight in the cleavage bonding liquid was 6000, and the remaining components were the same as those in preparation example 1.
Comparative preparation 7:
the polypropylene glycol molecular weight in the cleavage bonding liquid was 8000, and the remaining components were the same as those in preparation example 1.
Example 1: blood genomic DNA extraction
In this example, genomic DNA from different blood samples was extracted using the nucleic acid extraction or purification kit of preparation example 1 and comparative preparation examples 2 to 7, and the specific procedure was as follows:
(1) Sample preparation: 200 μl of whole blood sample was added to the centrifuge tube;
(2) Sample lysis: adding 500 mu L of the lysis binding solution provided in preparation example 1 and 20 mu L of magH1N Silica into the centrifuge tube in the step (1), uniformly mixing and incubating for 10min at the temperature of 65 ℃;
(3) Nucleic acid adsorption: carrying out magnetic separation by using a magnetic frame, discarding liquid after the magnetic beads are completely adsorbed, and obtaining the magnetic beads after adsorption;
(4) First cleaning: adding 500 mu L of a first general cleaning solution into the magnetic beads after adsorption obtained in the step (3), shaking and uniformly mixing for 1min, then placing the magnetic beads on a magnetic frame, magnetically separating until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain first cleaning magnetic beads;
(5) And (3) second cleaning: adding 500 mu L of a second general-purpose cleaning solution into the first cleaning magnetic beads obtained in the step (4), shaking and uniformly mixing for 1min, then placing the mixture on a magnetic frame, magnetically separating the mixture until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain second cleaning magnetic beads;
(6) Sample elution: adding 100 mu L of eluent into the second cleaning magnetic beads obtained in the step (5), shaking, incubating and eluting at 60 ℃ for 5min, placing the magnetic beads on a magnetic rack, collecting the DNA solution obtained after eluting after the magnetic beads are adsorbed, and storing the DNA solution at-80 ℃.
The nucleic acid concentration (Nanodrop) was measured after comparing the above-extracted 5 frozen EDTA anticoagulated whole blood samples, and the DNA extraction results of preparation example 1 and comparative preparation example were compared, and the measurement results are shown in table 1 below. As can be seen from the results of table 1, the rapid nucleic acid extraction kit of the present invention has significantly better extraction effect than the comparative preparation. Furthermore, the inventors observed that the DNA eluents extracted in comparative preparation 2 and comparative preparation 3 were not clear in color, and exhibited pale yellow or pale red, whereas the DNA eluate of preparation 1 was clear and colorless. Comparative preparation 1 and comparative example 1 found that when polypropylene glycol was not added to the lysate, DNA could not be extracted, i.e., DNA could not be precipitated. By way of preparation example 1, comparative example 1 and comparative examples 4 to 7, we found that the amount of DNA extracted correlated with the molecular weight of polypropylene glycol, and that the DNA concentration increased from 1000 to 4000, but the DNA concentration decreased from 4000 to 8000. Surprisingly, the present invention provides an optimum extraction of polypropylene glycol having a molecular weight of 4000.
TABLE 1 genomic DNA extraction test results of example 1 blood samples
| Sample of | Preparation example 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 |
| Blood 1 | 79.5 | 5.46 | 45.6 | 35.6 | 23.1 | 45.6 | 45.6 | 26.9 |
| Blood 2 | 145.1 | 6.58 | 68.5 | 85.4 | 45.6 | 69.8 | 56.5 | 32.5 |
| Blood 3 | 98.4 | 7.84 | 48.7 | 78.4 | 49.5 | 78.6 | 34.5 | 39.5 |
| Blood 4 | 65.4 | 12.31 | 32.1 | 45.6 | 48.8 | 46.5 | 23.6 | 34.6 |
| Blood 5 | 49.8 | 3.56 | 24.6 | 46.5 | 21.5 | 35.2 | 29.5 | 12.6 |
Example 2: genomic DNA extraction of frozen whole blood and clinical fresh whole blood samples
Experiments genomic DNA of frozen whole blood samples and fresh whole blood samples from clinical oncology patients were extracted using the rapid nucleic acid extraction kit and comparative kit of preparation example 1 (QIAamp DNA mini Kits, qiagen, cat # 51304), essentially the same procedure as in example 1, but using a 96-position automated nucleic acid extractor for automated extraction.
(1) Sample and reagent preparation: 200 μl of whole blood sample was added to the corresponding well in plate 1 in the 96-deep well plate.
(2) Nucleic acid extraction: and (3) putting the 96 deep hole sample plate with the sample and the reagent added in the previous step into a 96-bit nucleic acid extractor, putting into a 96-bit magnetic rod sleeve, and running a program.
After about 20 minutes the 96-deep well plate was removed and the eluted DNA from the 96-deep well plate in bay 8 was transferred to a clean (nuclease free) centrifuge tube and stored at-80 ℃.
The comparative kit was operated with reference to instructions of QIAamp DNAmini Kits, using 200 μl whole blood for extraction,
the eluate was eluted using 100. Mu.L.
The above-extracted 6 frozen whole blood and 6 fresh whole blood of clinical tumor patients were subjected to detection of nucleic acid concentration and purity, and compared with the results of nucleic acid extraction by the comparative kit QIAamp DNA mini Kits, the detection results are shown in Table 2 below. As can be seen from table 2, the rapid nucleic acid extraction kit of the present invention has significantly better extraction effect than the comparative inlet kit. The kit developed by the invention has high speed, can complete the full-automatic operation flow only by 20 minutes, and compared with the kit of Qiagen company, the kit of Qiagen company needs 50 minutes, and the centrifugal column method cannot complete the automatic high-flux operation. Surprisingly, the kit developed by the invention can still extract very high-quality DNA for clinical fresh whole blood, has a much better recovery effect than the kit of Qiagen company, can extract complete DNA for frozen whole blood or fresh whole blood of clinical tumor patients, has clear strips and complete main band as seen from a gel electrophoresis chart (figure 1). The kit of Qiagen company can extract complete DNA from frozen whole blood, has clear band, and has no clear main band for fresh whole blood of clinical tumor patients, and has low concentration and low purity.
TABLE 2 example 2 Whole blood sample DNA extraction test results
The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
It should be noted that the above embodiments can be freely combined as needed. The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A lysis conjugate, the lysis conjugate comprising: sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, a Gemini surfactant, polypropylene glycol and water.
2. The lysis conjugate of claim 1, wherein:
the lysis conjugate comprises: 50-500mM sodium maleate, 1-8M guanidine hydrochloride, 1-8M sodium perchlorate, 50-500mM ammonium chloride, 0.1-20% Brij 30, 0.2-20% Gemini surfactant, 5-30% polypropylene glycol and the balance water, and the pH of the lysis-binding solution is 5.0-9.0; wherein the concentration of Brij 30, gemini surfactant and polypropylene glycol is calculated by mass volume percent;
preferably, the lysis-binding fluid comprises: 50-200mM sodium maleate, 1-5M guanidine hydrochloride, 2-6M sodium perchlorate, 100-300mM ammonium chloride, 0.5-10% Brij 30, 0.5-10% Gemini surfactant, 10-25% polypropylene glycol and the balance water, and the pH of the lysis-binding solution is 5.5-8.5;
it is further preferred that the lysis binding fluid comprises: 75-175mM sodium maleate, 1.5-4.5M guanidine hydrochloride, 2-5M sodium perchlorate, 150-250mM ammonium chloride, 1-5% Brij 30, 1-8% Gemini surfactant, 12.5-22.5% polypropylene glycol and balance water, and the pH of the lysate is 7-8.5;
further and preferred, the lysis-binding fluid comprises 125mM sodium maleate, 4M guanidine hydrochloride, 5M sodium perchlorate, 200mM ammonium chloride, 2.5% Brij 30, 2.5% Gemini surfactant, 20% polypropylene glycol and the balance water, and the pH of the lysis-binding fluid is 8.5.
3. The lysis conjugate of claim 1 or 2, wherein:
the Gemini surfactant is selected from one or a combination of any more of octadecylamine polyoxyethylene ether biquaternary ammonium salt, cetyl polyoxyethylene ether dimethyl octyl ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride, ethylene bis (hexadecamide propyl dimethyl ammonium bromide), dodecyl polyoxyethylene ether dimethyl octyl ammonium chloride and dodecyl polyoxyethylene ether dimethyl dodecyl ammonium bromide;
preferably, the Gemini surfactant is dodecyl polyoxyethylene ether dimethyl octyl ammonium chloride.
4. A lysis binding fluid according to any of claims 1 to 3 wherein the polypropylene glycol has a molecular weight of 200-20000; preferably, the polypropylene glycol has a molecular weight of 500-10000; more preferably, the polypropylene glycol has a molecular weight of 1000 to 8000; it is further preferred that the polypropylene glycol molecular weight is 4000.
5. A nucleic acid extraction kit, characterized in that:
the nucleic acid kit comprises a reagent for a lysis binding solution for preparing the lysis binding solution according to any one of claims 1 to 4;
preferably, the reagent for the lysis binding fluid consists of the following components: (i) Sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, a Gemini surfactant, and polypropylene glycol; or (ii) sodium maleate, guanidine hydrochloride, sodium perchlorate, ammonium chloride, brij 30, gemini surfactant, polypropylene glycol and water for partitioning the components of (i) into the lysis binding fluid.
6. The kit of claim 5, wherein the nucleic acid extraction kit further comprises:
a reagent for preparing a magnetic bead suspension;
a cleaning liquid reagent for preparing a cleaning liquid; and/or
Eluent agent for preparing eluent.
7. The kit of claim 6, wherein:
the magnetic bead suspension comprises a dispersion liquid and 2-20% by mass and volume of superparamagnetic silicon particles with the particle diameter of 0.1-10 mu M, the pH value of the magnetic bead suspension is 4.0-7.0, and the dispersion liquid comprises 0.05-0.2M sodium chloride and 0.01-0.05% NaN 3 And the balance water, wherein the amount of superparamagnetic silicon particles in the magnetic bead suspension is in mass volume percent;
preferably, the magnetic bead suspension comprises a dispersion liquid and 5-10% by mass and volume of superparamagnetic silicon particles with the particle diameter of 0.5-5 μm, the pH of the magnetic bead suspension is 5.0-6.0, and the dispersion liquid comprises 0.05-0.2M sodium chloride and 0.01-0.05% NaN 3 And the balance water;
more preferably, the magnetic bead suspension comprises a dispersion and a mass volume percentage of 5% superparamagnetic silica particles having a particle size of 0.5-5 μm, the pH of the magnetic bead suspension is 5.0, the dispersion comprises 0.05M sodium chloride, 0.05% NaN 3 And the balance water.
8. The kit according to claim 6 or 7, wherein:
the cleaning liquid comprises a first general cleaning liquid and a second general cleaning liquid; the reagent for the cleaning liquid comprises a first cleaning test for preparing a first general cleaning liquid and a second cleaning reagent for preparing a second general cleaning liquid;
the first general cleaning solution comprises 5-1000mM Tris-HCl,10-1000mM lithium chloride and 1-20mM EDTA-Na 2 1-5M sodium perchlorate and 50-80% absolute ethanol, pH4-8; preferably, the first universal washing liquid is 10-500mM Tris-HCl,50-500mM lithium chloride, 2-10mM EDTA-Na 2 2-4M sodium perchlorate and 50-60% absolute ethanol, pH4-7; more preferably, the first universal washing solution comprises 100mM Tris-HCl,200mM lithium chloride, 2mM EDTA-Na 2 3M sodium perchlorate and 50% absolute ethanol, pH5.5;
the second general-purpose cleaning liquid comprises: 10-200mM Tris-HCl,10-100mM lithium chloride and 30-70% polypropylene glycol 400, pH5-8; preferably, the second universal washing solution comprises 20-100mM Tris-HCl,50-100mM lithium chloride and 40-60% polypropylene glycol 400, pH7; more preferably, the second universal washing solution comprises 50mM Tris-HCl,100mM lithium chloride and 50% polypropylene glycol 400, pH7; and/or
The eluate contained 10mM Tris-HCl and had a pH of 8.5.
9. A method for extracting or purifying nucleic acid from a biological sample, characterized in that the method employs the lysis conjugate of any one of claims 1 to 4 or the kit of any one of claims 5 to 8 for extraction or purification.
10. The method according to claim 9, characterized in that it comprises the steps of:
(1) Sample preparation: adding the biological sample into a centrifuge tube;
(2) Sample lysis: adding the lysis binding solution and the magnetic bead suspension into the centrifuge tube, uniformly mixing at the temperature of 40-80 ℃ and incubating for 5-30min;
(3) Nucleic acid adsorption: performing magnetic separation by using a magnetic frame, discarding supernatant obtained after the magnetic separation is completed, and obtaining magnetic beads after adsorption;
(4) First cleaning: adding a first general cleaning solution into the adsorbed magnetic beads, carrying out magnetic separation by using a magnetic frame after uniformly mixing, and then discarding the liquid obtained after the magnetic separation is completed to obtain first cleaning magnetic beads;
(5) And (3) second cleaning: adding a second general cleaning solution into the first cleaning magnetic beads, uniformly mixing, performing magnetic separation by using a magnetic rack, and discarding the liquid obtained after the magnetic separation is completed to obtain second cleaning magnetic beads;
(6) Sample elution: adding the eluent into the second cleaning magnetic beads, uniformly mixing, then shaking, incubating and eluting at 50-70 ℃ for 5-10min, and collecting the DNA solution obtained after eluting;
preferably, in step (1), the whole blood sample is used in a volume of 200 μl; in step (2), adding 1-3 times, preferably 2-3 times, the volume of the whole blood sample of the lysis buffer; in step (2), 10-30. Mu.L of the magnetic bead suspension is added; in step (4), 200 to 1000. Mu.L, more preferably 500 to 700. Mu.L, of the first universal cleaning solution is added; in step (5), 200 to 1000. Mu.L, more preferably 500 to 700. Mu.L, of the second universal cleaning solution is added; in step (6), 40-200. Mu.L, more preferably 50-100. Mu.L, of the eluent is added and the elution is incubated at 50-70℃for 3-6min, e.g.5 min, e.g.60 ℃.
11. The method according to claim 9 or 10, characterized in that:
the biological sample is a whole blood sample;
preferably, the biological sample is a frozen whole blood sample or a fresh whole blood sample such as a clinical fresh whole blood sample.
12. Use of a lysis binding fluid according to any of claims 1 to 4 or a kit according to any of claims 5 to 8 for nucleic acid extraction of a biological sample, in particular a whole blood sample, such as a frozen whole blood sample or a fresh whole blood sample, such as a clinical fresh whole blood sample.
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| CN118546922A (en) * | 2024-07-30 | 2024-08-27 | 恒昱生物制药(山东)有限公司 | A cosmetic DNA sodium raw material and its whitening and sunscreen application and preparation method |
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