CN116769767A - Nucleic acid extraction kit and nucleic acid extraction method - Google Patents
Nucleic acid extraction kit and nucleic acid extraction method Download PDFInfo
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- CN116769767A CN116769767A CN202310273784.4A CN202310273784A CN116769767A CN 116769767 A CN116769767 A CN 116769767A CN 202310273784 A CN202310273784 A CN 202310273784A CN 116769767 A CN116769767 A CN 116769767A
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 128
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 128
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 128
- 238000000605 extraction Methods 0.000 title claims abstract description 56
- 239000011324 bead Substances 0.000 claims abstract description 59
- 239000006166 lysate Substances 0.000 claims abstract description 33
- 239000003480 eluent Substances 0.000 claims abstract description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000005406 washing Methods 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 22
- 239000007983 Tris buffer Substances 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 20
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 20
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 19
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 19
- 239000000661 sodium alginate Substances 0.000 claims abstract description 19
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 19
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 15
- 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 abstract description 14
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 12
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims abstract description 12
- LBCZOTMMGHGTPH-UHFFFAOYSA-N 2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCO)C=C1 LBCZOTMMGHGTPH-UHFFFAOYSA-N 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000007853 buffer solution Substances 0.000 claims abstract description 10
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000008213 purified water Substances 0.000 claims abstract description 9
- 239000002904 solvent Substances 0.000 claims abstract description 9
- 229920001213 Polysorbate 20 Polymers 0.000 claims abstract description 8
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims abstract description 8
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 7
- 239000012472 biological sample Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 210000003296 saliva Anatomy 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 239000004593 Epoxy Substances 0.000 claims description 2
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 claims description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- 150000001299 aldehydes Chemical group 0.000 claims description 2
- 238000004945 emulsification Methods 0.000 claims description 2
- 210000003608 fece Anatomy 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 238000002955 isolation Methods 0.000 claims 7
- 238000002360 preparation method Methods 0.000 claims 1
- 230000003321 amplification Effects 0.000 description 13
- 238000003199 nucleic acid amplification method Methods 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 108010067770 Endopeptidase K Proteins 0.000 description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 239000002156 adsorbate Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 108010061100 Nucleoproteins Proteins 0.000 description 3
- 102000011931 Nucleoproteins Human genes 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 150000002357 guanidines Chemical class 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 230000009972 noncorrosive effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 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 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- -1 D30 nucleic acid Chemical class 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical group O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 238000010200 validation analysis Methods 0.000 description 1
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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
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- 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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Abstract
The invention discloses a nucleic acid extraction kit and a nucleic acid extraction method, comprising a lysate, magnetic beads and an eluent, wherein the lysate comprises the following components: tris buffer solution 20-100 mmol/L and pH value 5.5-7.5; 5-10 g/L NaCl; 2-10 g/L of sodium dodecyl sulfate; 5-10 g/L guanidine isothiocyanate; EDTA 5-10 g/L; BSA 1-5 g/L; 115 g/L to 5g/L; NP-40 2-5 g/L; tween 20 to 10g/L; the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads; the washing liquid comprises the following components: 50-100 mmol/L PBS buffer solution, and the pH value is 7.0-7.5; 60% -80% of ethanol; the eluent comprises the following components: 50-100 mmol/L Tris buffer solution and pH value of 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water. The kit has the advantages of simplicity, effectiveness, rapidness, convenience, short time consumption, high nucleic acid extraction efficiency and the like, and can meet the extraction requirements of a large number of samples.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a nucleic acid extraction kit and a nucleic acid extraction method.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Nucleic acid is a biological macromolecular compound polymerized from many nucleotides, and is one of the most basic substances in life. Nucleic acids are widely found in all animals, plant cells, microorganisms, in vivo nucleic acids and proteins to form nucleoproteins. Nucleic acid comprises DNA and RNA molecules, the molecules exist in a state of being combined with protein in cells, and the main steps of nucleic acid extraction comprise: lysing the cells; removing biological macromolecules such as proteins, polysaccharides, lipids and the like combined with the nucleic acid; removing other unwanted nucleic acid molecules; precipitating the nucleic acid to purify the nucleic acid; removing impurities such as salts. The procedures are complicated and the time consumption is long. Along with the rapid development of molecular biology in recent years, the application of PCR detection technology is more and more widespread, and the workload of nucleic acid extraction is rapidly increased, so that the traditional nucleic acid extraction mode cannot meet the requirements of detection of a large number of diseases.
In addition, proteinase K is generally contained in the conventional cell lysate, and the proteinase K requires a low temperature for transportation, which tends to cause inconvenience in transportation.
In addition, there are three types of cleavage modes currently in common use: physical, chemical and biological means. Among them, physical means are capable of sufficiently lysing cells, but may cause DNA fragmentation. Chemical means such as alkali cleavage, CTAB cleavage, etc. introduce toxic organic solvents, the operation is complicated and the extraction efficiency is low. The specific functional groups can be modified on the surface of the magnetic carrier by utilizing the nano magnetic beads, so that the reversible adsorption of the nucleic acid is realized, but the organic solvent is still added into the extraction kit of the current magnetic bead method, and is easy to remain in the prepared nucleic acid, and the reaction is easy to be inhibited when the PCR reaction is carried out, so that the subsequent PCR reaction is not facilitated.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a nucleic acid extraction kit and a nucleic acid extraction method. The kit has the advantages of simplicity, effectiveness, rapidness, convenience, short time consumption, high nucleic acid extraction efficiency and the like, and can meet the extraction requirements of a large number of samples.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a nucleic acid extraction kit comprising a lysate, magnetic beads, a wash solution, and an eluent, wherein,
the components of the lysate are as follows: tris buffer solution 20-100 mmol/L and pH value 5.5-7.5; 5-10 g/L NaCl; 2-10 g/L of sodium dodecyl sulfate; 5-10 g/L guanidine isothiocyanate; EDTA 5-10 g/L; BSA 1-5 g/L; 115 g/L to 5g/L; NP-40 2-5 g/L; tween 20 to 10g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
superparamagnetic Fe by emulsification method 3 O 4 The nano particles are directly coated in crosslinked sodium alginate to obtain superparamagnetic sodium alginate nano magnetic beads with the particle size of 25-30nm, and then ethylene oxide and glutaraldehyde are used for activating and modifying the magnetic sodium alginate to ensure that the surfaces of the magnetic sodium alginate have epoxy functional groups and aldehyde functional groups respectively, so that the activated carrier can fix nucleic acid and release nucleic acid under different pH values through a covalent coupling method.
The washing liquid comprises the following components: 50-100 mmol/L PBS buffer solution, and the pH value is 7.0-7.5; 60% -80% of ethanol, wherein the% refers to volume percentage;
the eluent comprises the following components: 50-100 mmol/L Tris buffer solution and pH value of 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
The lysate can denature the protein, can rapidly dissolve the protein, causes the cell structure to be broken, and the secondary structure of the nucleoprotein is destroyed so as to separate the nucleoprotein from the nucleic acid. The effect of the lysate is to release the nucleic acid from the intracellular feed. In addition to nucleic acid, the lysed cells have a number of salts, which require washing solutions to precipitate the nucleic acid, which may otherwise interfere with the results of subsequent experiments. The eluting solution may elute the nucleic acid from the magnetic beads.
In the lysate, tris-HCL buffer solution can provide a proper cracking environment, naCL plays an important role in maintaining the stability of a nucleic acid structure, EDTA can inhibit the damage of nuclease in a sample to the nucleic acid and has an important role in maintaining the stability of the nuclease, and sodium dodecyl sulfate, guanidine isothiocyanate and NP-40 can denature proteins and destroy cell membrane structures so as to separate the proteins from the nucleic acid, so that free nucleic acid can be obtained in a cracking system, and the raw materials cooperate with each other to remove impurities and provide a stable environment for the nucleic acid.
The magnetic beads are small beads which are formed by coating a ferroferric oxide core by a certain tissue, can be adsorbed by a magnet and can adsorb nucleic acid through a surface coating, and the effect of adsorbing nucleic acid by the magnetic beads is good, so that the extraction rate of the nucleic acid is high.
In the washing liquid, if too little ethanol is added, the washing is not clean, impurities can appear, and if too much ethanol is added, the ethanol is not volatilized thoroughly, and a certain inhibition effect can be generated on the subsequent amplification.
In a second aspect, the present invention provides a nucleic acid extraction method using the nucleic acid extraction kit, comprising the steps of:
placing a biological sample into a mixed solution of magnetic beads and a lysate, vibrating and uniformly mixing, adsorbing the lysed nucleic acid on the magnetic beads, and adsorbing the nucleic acid on the magnetic beads on the magnetic bar sleeve through the magnetic force of the magnetic bar sleeve;
transferring the stirring sleeve into a washing liquid, and removing impurities;
the DNA is then purified in an eluent to obtain a nucleic acid sample.
In some embodiments, the biological sample is saliva, a pharyngeal swab, serum, or stool.
In some embodiments, the shaking-mixing time is 8-12s, preferably 10s.
The specific operation process is as follows:
(1) Taking out three reagent plates of the lysate, the washing solution and the eluent, lightly throwing for 2-3 times, and throwing the liquid hung on the hole wall to the bottom of the reagent plate.
(2) And (3) tearing off the aluminum foil sealing films of the three plates to prevent liquid from splashing.
(3) 200. Mu.L of sample was added to plate 1 (lysate)
(4) Placing the three reagent plates and the stirring sleeve at corresponding positions of a nucleic acid extractor
(5) Selecting a nucleic acid extraction program and running
(6) And after the program is finished, taking out the reagent plate and the stirring sleeve, wherein the eluent is the extracted nucleic acid sample.
The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:
1. the nucleic acid extraction kit can rapidly separate and purify nucleic acid samples from samples such as swabs, serum and saliva, and the whole process only comprises three steps of cracking, washing and eluting, so that the kit is relatively rapid, efficient and time-saving; the lysate of the nucleic acid extraction kit does not contain proteinase K, and the transport inconvenience is reduced because proteinase K needs to be at a low temperature during transport.
2. The lysate of the nucleic acid extraction kit does not contain toxic reagents, can fully release nucleic acid, and has the advantages of higher concentration and purity of the obtained nucleic acid, low cost and no pollution.
The nano magnetic beads in the nucleic acid extraction kit can adsorb or release nucleic acid in a specific pH value solution through specific sodium alginate modification, and an organic solvent and an amplification inhibitor are not needed in the extraction process, so that the obtained nucleic acid has higher quality.
The eluent does not contain any organic solvent, and the extracted nucleic acid solution can be directly applied to the subsequent PCR amplification reaction, so that the experimental steps are greatly simplified, and the time cost and the material cost are shortened. Can meet the requirements of hospitals and detection institutions on the nucleic acid extraction efficiency and quality.
The nucleic acid extraction kit is simple and convenient to operate, has short time consumption, can be used for manual scientific research operation, is also suitable for a nucleic acid extraction instrument, has safe and nontoxic reagent components, has no toxic or harmful effect on human bodies and environment, and can be stored at normal temperature.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a nucleic acid amplification curve extracted by a magnetic bead nucleic acid extraction kit of a first control group according to the present invention;
FIG. 2 is a nucleic acid amplification curve extracted by the magnetic bead nucleic acid extraction kit of example 1 of the present invention;
FIG. 3 is a nucleic acid amplification curve extracted by the magnetic bead nucleic acid extraction kit of example 2 of the present invention;
FIG. 4 is a nucleic acid amplification curve extracted by the magnetic bead nucleic acid extraction kit of example 3 of the present invention;
FIG. 5 is a nucleic acid amplification curve extracted by the magnetic bead nucleic acid extraction kit of example 4 of the present invention;
FIG. 6 is a nucleic acid amplification curve extracted by the magnetic bead nucleic acid extraction kit of example 5 of the present invention;
FIG. 7 is a nucleic acid amplification curve extracted by the magnetic bead nucleic acid extraction kit of example 6 of the present invention;
FIG. 8 is a graph showing the amplification of nucleic acid extracted by the magnetic bead nucleic acid extraction kit of the second control group.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention is further illustrated below in connection with comparative examples and examples.
Comparative example 1
A nucleic acid extraction kit comprises a lysate, proteinase K, a nucleic acid adsorbate, a cleaning solution and an eluent.
Lysate:
guanidine salt (m/v) 50% ± 5%;
NaCl·············1％±0.5％(m/v)；
EDTA·············0.2％±0.05(m/v)；
proteinase K & ltS & gt & lt/S & gt 10mg/ml-20mg/ml;
the magnetic beads are Fe 3 O 4 Particle size is 200nm;
nucleic acid adsorbate:
isopropyl alcohol 20-30%;
cleaning liquid: ultrapure water.
Eluent: DEPC water.
Example 1
The magnetic bead method nucleic acid extraction kit of this embodiment includes: lysate, magnetic beads, washing liquid and eluent, wherein:
the components of the lysate are as follows: tris buffer 20mmol/L, pH 5.5; naCl5g/L; 2g/L of sodium dodecyl sulfate; 5g/L guanidine isothiocyanate; EDTA 5g/L; BSA 1g/L; 115 g/L of Jimei; NP-40 2g/L; tween 20 g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: 50mmol/L PBS buffer solution with pH value of 7.0; 60% of ethanol;
the eluent comprises the following components: 50mmol/L Tris buffer, pH 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
The application method of the magnetic bead method nucleic acid extraction kit comprises the following steps:
a. adding a biological sample into a lysis solution (containing magnetic beads) plate, vibrating and uniformly mixing for 10s, performing instantaneous centrifugation, adsorbing the lysed nucleic acid on the magnetic beads at the moment, and adsorbing the nucleic acid on a stirring sleeve under the action of a binding agent;
b. the stirring sleeve is moved into the washing liquid, and impurities such as protein, sugar, RNA and the like can be removed in the step;
c. the DNA is then purified in an eluent to obtain a nucleic acid sample.
The biological sample comprises saliva, pharyngeal swab, serum, feces and other relevant samples.
Example 2
The magnetic bead method nucleic acid extraction kit of this embodiment includes: lysate, magnetic beads, washing liquid and eluent, wherein:
the components of the lysate are as follows: tris buffer 40mmol/L, pH 6.0; naCl7g/L; 5g/L of sodium dodecyl sulfate; 6g/L guanidine isothiocyanate; EDTA 6g/L; BSA 2g/L; 115 g/L of Jimei; NP-40 3g/L; tween 20 g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: 60mmol/L PBS buffer solution with pH value of 7.27; 65% of ethanol;
the eluent comprises the following components: 60mmol/L Tris buffer and pH value of 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
The detection method is the same as that of example 1.
Example 3
The magnetic bead method nucleic acid extraction kit of this embodiment includes: lysate, magnetic beads, washing liquid and eluent, wherein:
the components of the lysate are as follows: 60mmol/L Tris buffer and pH value of 6.5; naCl7g/L; 4g/L of sodium dodecyl sulfate; guanidine isothiocyanate 7g/L; EDTA 7g/L; BSA 3g/L; 115 g/L of Jimei; NP-40 3g/L; tween 20 g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: PBS buffer solution with the concentration of 65mmol/L and the pH value of 7.0; 65% of ethanol;
the eluent comprises the following components: 50-100 mmol/L Tris buffer solution and pH value of 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
The detection method is the same as that of example 1.
The nucleic acids extracted in examples 1 to 3 were measured for concentration and purity using an eppendorf D30 nucleic acid protein meter, wherein the purity of the nucleic acids was the ratio of A260 to A280, A260 represents the absorbance of the nucleic acids at a wavelength of 260nm, and A280 represents the absorbance of the nucleic acids at a wavelength of 280nm, and the results are shown in Table 1.
TABLE 1 results of the concentration and purity of salivary nucleic acids extracted in different examples
| Sample source | Concentration (ng/. Mu.L) | Purity (A260/A280) |
| Comparative example 1 | 43.5 | 1.83 |
| Example 1 | 44.2 | 1.85 |
| Example 2 | 45.7 | 1.88 |
| Example 3 | 46.3 | 1.90 |
As can be seen from Table 1, the purity of the nucleic acid extracted from the kit of the invention is better than that of the nucleic acid extracted from the kit of the control group, the ratio of the high-quality nucleic acid A260/A280 is in the range of 1.8-2.0, and the purity of the nucleic acid extracted from the kit of the invention is in the range of 1.8-2.0, which indicates that the nucleic acid has no pollution such as protein and the like, and the purity is higher, and can be used for the next step.
After the completion of the extraction, the sialic acid DNA was used as a template, and amplified by a fluorescent quantitative PCR amplification apparatus, and the amplification results of comparative example 1 and examples 1 to 3 are shown in FIG. 1.
As can be seen from FIG. 1, the amplification curves of comparative example 1 and examples 1-3 are identical and the CT values are also close, indicating that the concentrations are substantially identical, so that the kit of the invention is completed to meet the requirements for nucleic acid extraction.
Example 4
A nucleic acid extraction kit, which comprises a lysate, magnetic beads, a washing solution and an eluent;
the components of the lysate are as follows: 80mmol/L Tris buffer and pH value of 7.0; naCl8g/L; 8g/L of sodium dodecyl sulfate; 8g/L guanidine isothiocyanate; EDTA 9g/L; BSA 4g/L; 115 g/L of Jimei; NP-40 4g/L; tween 20 g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: 70mmol/L PBS buffer solution with pH value of 7.2; 75% of ethanol;
the eluent comprises the following components: 80mmol/L Tris buffer and pH value of 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
Example 5
A nucleic acid extraction kit, which comprises a lysate, magnetic beads, a washing solution and an eluent;
the components of the lysate are as follows: tris buffer 75mmol/L, pH 6.8; naCl8.5g/L; 8g/L of sodium dodecyl sulfate; 7.5g/L guanidine isothiocyanate; EDTA 8.5g/L; BSA 3.5g/L; very beautiful 115.5 g/L; NP-40.5 g/L; tween 20.5 g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: PBS buffer solution 75mmol/L, pH value 6.8; 68% of ethanol;
the eluent comprises the following components: tris buffer 75mmol/L, pH 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
Example 6
A nucleic acid extraction kit, which comprises a lysate, magnetic beads, a washing solution and an eluent;
the components of the lysate are as follows: tris buffer 90mmol/L, pH 7.5; naCl10g/L; 10g/L of sodium dodecyl sulfate; 10g/L guanidine isothiocyanate; EDTA 10g/L; BSA 5g/L; 115 g/L of Jimei; NP-40 g/L; tween 20 10g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: 100mmol/L PBS buffer solution with pH value of 7.5; 80% of ethanol;
the eluent comprises the following components: 100mmol/L Tris buffer and pH 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
Comparative example 2
A nucleic acid extraction kit comprises a lysate, proteinase K, a nucleic acid adsorbate, a cleaning solution and an eluent.
The composition of the lysate is:
guanidine salt (50% + -5%, m/v);
NaCl············1％±0.5％，m/v；
EDTA············0.2％±0.05％，m/v；
proteinase K & ltS & gt & lt/S & gt & ltS & gt 10mg/ml-20mg/ml;
the magnetic beads are Fe 3 O 4 The particle size was 200nm.
The nucleic acid adsorbate is 20-30% isopropyl alcohol;
cleaning liquid: ultrapure water;
eluent: DEPC water.
Determination of nucleic acid concentration and purity in saliva extracted in comparative example 2 and example 4, example 5 and example 6 using nanodrop micro-spectrophotometer with purity A 260 /A 280 Ratio of A 260 Representing the absorbance value of the nucleic acid at 260nm, A 280 The absorbance values of the nucleic acids at 280nm are shown and the test results are shown in Table 2.
TABLE 2 concentration and purity of nucleic acids in saliva samples
As can be seen from Table 2, the concentrations and purities of the nucleic acids obtained in example 4, example 5 and example 6 are higher than those in comparative example 2, indicating that the nucleic acid extraction kit of the present invention can obtain high quality nucleic acids, wherein the concentration and purity of the nucleic acids extracted in example 5 are higher than those in example 4 and example 6.
The stability of the kit was verified by placing comparative example 2 and example 2 in a non-corrosive gas-free, light-tight environment at room temperature. The same samples were assayed three times per month for comparative example 2 and example 5, and the average of Ct values was taken and the specific data is shown in table 3.
TABLE 3 stability validation results
As is clear from the data in Table 3, the nucleic acid extraction kit of the present invention can be stored stably for 15 months in the presence of a non-corrosive gas in the absence of light, whereas the kit of comparative example 2 can be stored for 12 months, and the stability is drastically lowered at 13 months.
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 (8)
1. A nucleic acid extraction kit, characterized in that: comprises a lysate, magnetic beads, a washing solution and an eluent, wherein,
the components of the lysate are as follows: tris buffer solution 20-100 mmol/L and pH value 5.5-7.5; 5-10 g/L NaCl; 2-10 g/L of sodium dodecyl sulfate; 5-10 g/L guanidine isothiocyanate; EDTA 5-10 g/L; BSA 1-5 g/L; 115 g/L to 5g/L; NP-40 2-5 g/L; tween 20 to 10g/L;
the magnetic beads are superparamagnetic sodium alginate nanometer magnetic beads;
the washing liquid comprises the following components: 50-100 mmol/L PBS buffer solution, and the pH value is 7.0-7.5; 60% -80% of ethanol;
the eluent comprises the following components: 50-100 mmol/L Tris buffer solution and pH value of 7.5; EGTA 5g/L; sodium azide 0.5g/L; the solvent is purified water.
2. The nucleic acid extraction kit of claim 1, wherein: the components of the lysate are as follows: 30-90 mmol/L Tris buffer solution and pH value of 6-7.5; 5-10 g/L NaCl; 5-10 g/L of sodium dodecyl sulfate; 5-10 g/L guanidine isothiocyanate; EDTA 5-10 g/L; BSA 2-5 g/L; 115 g/L to 5g/L; NP-40 2-5 g/L; tween 20-10 g/L.
3. The nucleic acid extraction kit of claim 2, wherein: the components of the lysate are as follows: 50-70 mmol/L Tris buffer solution and pH value of 6-7; 5-10 g/L NaCl; 5-10 g/L of sodium dodecyl sulfate; 5-10 g/L guanidine isothiocyanate; EDTA 5-10 g/L; BSA 2-5 g/L; 115 g/L to 5g/L; NP-40 2-5 g/L; tween 20-10 g/L.
4. The nucleic acid extraction kit of claim 2, wherein: the preparation method of the superparamagnetic sodium alginate nanometer magnetic beads comprises the following steps: superparamagnetic Fe by emulsification method 3 O 4 The nano particles are directly coated in crosslinked sodium alginate, so that superparamagnetic sodium alginate nano magnetic beads with the particle size of 25-30nm can be obtained, and then ethylene oxide and glutaraldehyde are used for activating and modifying the magnetic sodium alginate, so that the surfaces of the magnetic sodium alginate have epoxy functional groups and aldehyde functional groups respectively.
5. A method for nucleic acid isolation using the nucleic acid isolation kit according to any one of claims 1 to 4, wherein: the method comprises the following steps:
placing a biological sample into a mixed solution of magnetic beads and a lysate, vibrating and uniformly mixing, adsorbing the lysed nucleic acid on the magnetic beads, and adsorbing the nucleic acid on the magnetic beads on the magnetic bar sleeve through the magnetic force of the magnetic bar sleeve;
transferring the stirring sleeve into a washing liquid, and removing impurities;
the DNA is then purified in an eluent to obtain a nucleic acid sample.
6. The method for nucleic acid isolation using the nucleic acid isolation kit according to claim 5, wherein: the biological sample is saliva, pharyngeal swab, serum or feces.
7. The method for nucleic acid isolation using the nucleic acid isolation kit according to claim 5, wherein: the shaking and mixing time is 8-12s.
8. The method for nucleic acid isolation according to claim 7, wherein: the shaking and mixing time is 10s.
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