CN114634925A - Nucleic acid extraction kit and method for extracting nucleic acid - Google Patents
Nucleic acid extraction kit and method for extracting nucleic acid Download PDFInfo
- Publication number
- CN114634925A CN114634925A CN202011480855.0A CN202011480855A CN114634925A CN 114634925 A CN114634925 A CN 114634925A CN 202011480855 A CN202011480855 A CN 202011480855A CN 114634925 A CN114634925 A CN 114634925A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- proteinase
- solution
- magnetic beads
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 92
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 92
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 92
- 238000000605 extraction Methods 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 86
- 239000011324 bead Substances 0.000 claims abstract description 74
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 67
- 239000000843 powder Substances 0.000 claims abstract description 35
- 239000007853 buffer solution Substances 0.000 claims abstract description 29
- 239000012139 lysis buffer Substances 0.000 claims abstract description 28
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000001179 sorption measurement Methods 0.000 claims abstract description 26
- 239000003480 eluent Substances 0.000 claims abstract description 18
- 239000003755 preservative agent Substances 0.000 claims abstract description 14
- 230000002335 preservative effect Effects 0.000 claims abstract description 14
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 6
- 229920002307 Dextran Polymers 0.000 claims abstract description 6
- 229930195725 Mannitol Natural products 0.000 claims abstract description 6
- 239000000594 mannitol Substances 0.000 claims abstract description 6
- 235000010355 mannitol Nutrition 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 239000003599 detergent Substances 0.000 claims description 21
- 239000000872 buffer Substances 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 10
- 239000010703 silicon Substances 0.000 claims description 9
- 229910052710 silicon Inorganic materials 0.000 claims description 9
- 239000003381 stabilizer Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 7
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 5
- -1 ethyl phenyl Chemical group 0.000 claims description 5
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 230000002421 anti-septic effect Effects 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical group CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 230000007547 defect Effects 0.000 abstract description 5
- 239000002552 dosage form Substances 0.000 abstract description 2
- 238000004108 freeze drying Methods 0.000 description 15
- 102000053602 DNA Human genes 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 239000000126 substance Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 102000006382 Ribonucleases Human genes 0.000 description 6
- 108010083644 Ribonucleases Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000005336 cracking Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 102000016911 Deoxyribonucleases Human genes 0.000 description 4
- 108010053770 Deoxyribonucleases Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 150000005846 sugar alcohols Chemical class 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 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 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 229920001612 Hydroxyethyl starch Polymers 0.000 description 2
- 229920002774 Maltodextrin Polymers 0.000 description 2
- 239000005913 Maltodextrin Substances 0.000 description 2
- 102000011931 Nucleoproteins Human genes 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000004067 bulking agent Substances 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229940050526 hydroxyethylstarch Drugs 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000000845 maltitol Substances 0.000 description 2
- 235000010449 maltitol Nutrition 0.000 description 2
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 description 2
- 229940035436 maltitol Drugs 0.000 description 2
- 229940035034 maltodextrin Drugs 0.000 description 2
- 229960001855 mannitol Drugs 0.000 description 2
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 229960002920 sorbitol Drugs 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 239000000811 xylitol Substances 0.000 description 2
- 235000010447 xylitol Nutrition 0.000 description 2
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 2
- 229960002675 xylitol Drugs 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- NRTLIYOWLVMQBO-UHFFFAOYSA-N 5-chloro-1,3-dimethyl-N-(1,1,3-trimethyl-1,3-dihydro-2-benzofuran-4-yl)pyrazole-4-carboxamide Chemical compound C=12C(C)OC(C)(C)C2=CC=CC=1NC(=O)C=1C(C)=NN(C)C=1Cl NRTLIYOWLVMQBO-UHFFFAOYSA-N 0.000 description 1
- ICZWAZVKLACMKR-CIUDSAMLSA-N Asp-His-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 ICZWAZVKLACMKR-CIUDSAMLSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241001523956 Parengyodontium album Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 229940066758 endopeptidases Drugs 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 125000005372 silanol group Chemical group 0.000 description 1
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Substances [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the technical field of biology, and particularly discloses a nucleic acid extraction kit and a method for extracting nucleic acid, wherein the nucleic acid extraction kit comprises: lysis buffer solution, proteinase K freeze-dried powder, rinsing buffer solution, eluent and magnetic bead adsorption solution; the proteinase K freeze-dried powder comprises: MOPSO, proteinase K, preservative, mannitol and dextran. By the mode, the dosage form of the proteinase K in the nucleic acid extraction kit is the freeze-dried powder, and the proteinase K can be stably stored at room temperature for a long time, so that the defects of the existing proteinase K solution are overcome.
Description
Technical Field
The application relates to the technical field of biology, in particular to a nucleic acid extraction kit and a method for extracting nucleic acid.
Background
The technology of separating and purifying nucleic acid is a basic technology of molecular biology experiments, and the nucleic acid extraction can not be separated from the initial target gene extraction, PCR product purification or 'glue recovery', plasmid extraction in gene cloning and other experimental processes.
In the prior art, proteinase K is commonly used as a catalyst for nucleic acid extraction, but a proteinase K solution needs to be stored at a low temperature of-20 ℃.
However, the inventor of the present application finds that, in a long-term research and development process, a proteinase K solution is colorless and transparent, and precipitates are generated, that is, the proteinase K solution is ineffective, if the kit is frequently opened to check the state of the proteinase K solution, heat exchange between air in the kit and the outside is caused, the activity of proteinase K is remarkably reduced, and the requirement of long-term stable storage is difficult to achieve.
Disclosure of Invention
Based on this, the application provides a nucleic acid extraction kit and a method for extracting nucleic acid, and the dosage form of the proteinase K in the nucleic acid extraction kit is freeze-dried powder which can be stably stored at room temperature for a long time, so that the defects of the existing proteinase K solution are overcome.
In order to solve the technical problem, the application adopts a technical scheme that: provided is a nucleic acid extraction kit comprising: lysis buffer solution, proteinase K freeze-dried powder, rinsing buffer solution, eluent and magnetic bead adsorption solution; the proteinase K freeze-dried powder comprises: MOPSO, proteinase K, preservative, mannitol and dextran.
In order to solve the above technical problem, another technical solution adopted by the present application is: there is provided a method for extracting nucleic acid based on the aforementioned nucleic acid extraction kit, comprising the steps of: mixing the lysis buffer solution, the proteinase K freeze-dried powder and the magnetic bead adsorption solution with a sample so as to obtain magnetic beads with adsorbed nucleic acids; washing the magnetic beads adsorbed with the nucleic acid by using a rinsing buffer solution, carrying out solid-liquid separation after washing, and collecting the magnetic beads adsorbed with the nucleic acid; and (3) eluting the washed magnetic beads adsorbed with the nucleic acid by using an eluent, carrying out solid-liquid separation after elution, and collecting supernatant so as to obtain a nucleic acid solution.
The beneficial effect of this application is: different from the condition of the prior art, the nucleic acid extraction kit comprises a lysis buffer solution, proteinase K freeze-dried powder, a rinsing buffer solution, an eluent and a magnetic bead adsorption solution, wherein the proteinase K freeze-dried powder can be stably stored at room temperature for a long time, so that the stability of the preparation is improved, and the defect that the proteinase K activity is obviously reduced due to frequent opening of the kit when the proteinase K solution is stored at-20 ℃ in a freezing way in the prior art is overcome. When the protease K freeze-dried powder is used, the protease K freeze-dried powder is dissolved in a lysis buffer solution and a magnetic bead adsorption solution, and the operation is convenient.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic flow chart of a method for extracting nucleic acid according to an embodiment of the present disclosure.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The present application provides a nucleic acid extraction kit, comprising: lysis buffer solution, proteinase K freeze-dried powder, rinsing buffer solution, eluent and magnetic bead adsorption solution. The proteinase K freeze-dried powder comprises: MOPSO, proteinase K, preservative, mannitol and dextran.
In particular, proteinase K of the present application is proteinase K (e.c.3.4.21.64), an extracellular endopeptide 3969224 enzyme synthesized by the fungus Tritirachium album Limber, which is a member of the serine protease class with the typical catalytic triad Asp-His-Ser, and is capable of cleaving the carboxyl-terminal peptide bond of aliphatic and aromatic amino acids, serving to disrupt cells and release nucleic acids. In addition, proteinase K degrades proteins bound to nucleic acids, facilitating the isolation of nucleic acids.
Proteinase K has a specific activity of more than 30U/mg, is one of the most active of the known endopeptidases, and non-specifically hydrolyses native and denatured proteins. Mature proteinase K consists of 279 amino acids, two disulfide bridges and two bonded calcium ions making the compact structure stable. Proteinase K has high stability at high temperatures (up to 65 ℃) and a wide pH range (pH 7.5-12.0). Its activity is increased in the presence of denaturants such as urea or SDS. The above properties make proteinase K very useful in biotechnological fields where non-specific protein degradation is required, in particular in the isolation of nucleic acids (DNA or RNA) from crude extracts and in the preparation of samples for DNA analysis, and in addition proteinase K can also be used in protein analysis fields, such as structure-based analysis.
The buffer solution of 3- (N-morpholine) -2-hydroxypropanesulfonic acid (MOPSO) is a nonionic buffer solution, the pH range of the buffer solution is 6.2-7.6, and the activity of RNase and DNase can be effectively inhibited.
The stabilizer can act as a freeze-dried matrix, which helps to maintain proteinase K activity, especially during long-term storage at temperatures of 4-30 ℃.
In another embodiment, the stabilizer may further comprise at least one filler, which may include dextran, maltodextrin, polyvinylpyrrolidone, hydroxyethyl starch, or a combination thereof.
In certain embodiments, the stabilizer may further comprise at least one sugar alcohol, which may include sorbitol, mannitol, xylitol, maltitol, or a combination thereof.
In another embodiment, the stabilizing agent may further comprise at least one non-reducing oligosaccharide, which may include trehalose, sucrose, raffinose, or combinations thereof.
The present application also provides stabilizers further comprising at least one bulking agent and at least one sugar alcohol, wherein the sugar alcohol can include the bulking agent can include dextran, maltodextrin, polyvinylpyrrolidone, hydroxyethyl starch, or a combination thereof, and the sugar alcohol can include sorbitol, mannitol, xylitol, maltitol, or a combination thereof.
The preservative may include NaN3Or ProClin300 or a combination thereof.
The proteinase K freeze-dried powder can be obtained by freeze-drying a proteinase K solution. Specifically, MOPSO buffer solution, proteinase K, preservative and stabilizer are transferred into a freeze-drying bottle, the freeze-drying bottle is placed in a low-temperature ethanol bath below-20 ℃ and rotated to ensure that solute is coated on the wall of the bottle as thin and uniform as possible, and after the solution is evaporated to dryness, the freeze-drying bottle is firmly fixed on a freeze dryer for 10 hours; taking down the freeze-drying bottle with the freeze-drying powder on the inner wall, vacuumizing, sealing, and storing at 4-30 deg.C.
Different from the condition of the prior art, the nucleic acid extraction kit comprises a lysis buffer solution, a proteinase K freeze-dried powder, a rinsing buffer solution, an eluent and a magnetic bead adsorption solution, wherein the proteinase K freeze-dried powder can be stably stored at room temperature for a long time, so that the stability of the preparation is improved, and the defect that the activity of proteinase K is obviously reduced due to frequent opening of the kit when the proteinase K solution is stored at-20 ℃ in a freezing way in the prior art is overcome. When the protease K freeze-dried powder is used, the protease K freeze-dried powder is dissolved in a lysis buffer solution and a magnetic bead adsorption solution, and the operation is convenient.
In one embodiment, the storage temperature of the nucleic acid extraction kit is 4-30 ℃.
Specifically, the proteinase K freeze-dried powder can be stably stored at 4-30 ℃ for a long time, and other solutions in the nucleic acid extraction kit can be stably stored at room temperature for a long time, so that the nucleic acid extraction kit can be stored in an environment of 4-30 ℃.
In one embodiment, the lysis buffer comprises: MOPSO buffer, guanidine isothiocyanate, nonionic detergent, anionic detergent, dithiothreitol, EDTA, isopropanol, sodium chloride and preservative. The rinse buffer included: sodium chloride and ethanol. The eluent comprises: RNase free water. The magnetic bead adsorption solution comprises: 0.5-1.5 μm magnetic bead, 40-300nm magnetic bead, MOPSO buffer solution, and antiseptic.
The function and action of each component in the cracking adsorption solution are as follows:
(1) the MOPSO buffer solution is a non-ionic buffer solution, the pH range of the MOPSO buffer solution is 6.2-7.6, and the MOPSO buffer solution can effectively inhibit the activities of RNase and DNase;
(2) taking the example of extracting RNA, most RNA in cells exists in the form of a nucleoprotein complex, and guanidinium isothiocyanate is a strong protein denaturant and can crack cells, promote the dissociation of the nucleoprotein complex, separate RNA from protein and release the RNA into a solution;
(3) the nonionic detergent and the anionic detergent can dissolve lipid, prevent the interference of the lipid on nucleic acid adsorption, have the function of promoting the formation of hydrogen bonds, improve the cell nucleus cracking capability, promote the release of DNA (deoxyribonucleic acid) and achieve strong cracking function without heating at room temperature;
wherein, the nonionic detergent can be ethyl phenyl polyethylene glycol (Nonidet P40, NP-40 for short), NP-40 is a very mild nonionic detergent, 1% concentration can basically destroy cell membranes, but the destruction effect on nuclear membranes is weak, the binding force with protein is strong, and the nonionic detergent can be used for dissolving membrane proteins under the non-denaturing condition;
the anionic detergent is Sodium Dodecyl Sulfate (SDS), and can crack cells under the condition of high temperature (55-65 ℃), so that chromosomes are isolated, proteins are denatured, and nucleic acid is rapidly released;
(4) EDTA is used for chelating divalent metal ions, thereby further inhibiting the degradation of DNA by DNA deoxyribonuclease, protecting DNA after the DNA is released into a lysis buffer solution, and the combination of EDTA and Tris can be used as a stabilizer of the whole lysis buffer solution to stabilize the pH value of the lysis buffer solution and an ion-free environment;
(5) dithiothreitol (DTT) is used in combination with EDTA to destroy disulfide bonds in RNase protein, denature RNase, inhibit phosphodiester bond cleavage caused by phenol oxidation and cross-linking of RNA and DNA, separate DNA from RNA, and effectively inhibit the activity of RNase and DNase; the high-concentration DTT is added into the lysis buffer solution to denature protein, and under the coordination of the guanidinium isothiocyanate, the high-concentration guanidinium isothiocyanate can better precipitate nucleic acid and fully denature the protein, so that the nucleic acid released into the lysis buffer solution is well separated from the protein, the nucleic acid is fully released into the lysis buffer solution, and the extraction efficiency of microbial nucleic acid is improved;
(6) sodium chloride supplies Na to the cationic bridge between the phosphate group and the silanol group of nucleic acids+Ions further enhance the adsorption of the nucleic acid and silicon-based or silicon-hydroxyl on the surface of the magnetic beads, and are beneficial to the specific combination of the nucleic acid and the magnetic beads；
(7) The isopropanol can act together with detergent to precipitate and denature protein.
Function and action of the components in the rinse buffer:
(1) the sodium chloride with a certain concentration can ensure that the nucleic acid is not dissolved in the aqueous solution, increase the precipitation of the nucleic acid, keep the stability of the nucleic acid, prevent the nucleic acid from being damaged by DNAse and RNAse and better play a role in rinsing the DNA;
(2) ethanol can eliminate the hydration layer of nucleic acid to expose negatively charged phosphate groups, and Na provided by sodium chloride+Such counter ions can bind to negatively charged phosphate groups and reduce the repulsion between polynucleotide chains at the site of precipitate formation, so that the combined action of sodium chloride and ethanol facilitates the collection of nucleic acids on magnetic beads and also serves to wash impurities.
The preparation method of the RNase-free water comprises the following steps: DEPC (diethylpyrocarbonate) was added to deionized water to a final concentration of 0.05% (V/V), and after standing at room temperature (22-25 ℃) for 10-12 hours, DEPC was removed at 121 ℃ for 20 minutes under high pressure and left at room temperature for further use.
By the mode, guanidine isothiocyanate is combined with a nonionic detergent and an anionic detergent, so that the aim of quickly cracking cells at room temperature can be fulfilled. Meanwhile, only one-step rinsing is needed due to the reduction of the salt ion concentration.
In one embodiment, the MOPSO buffer has a concentration of 10-100mM (e.g., 10mM, 20mM, 30mM, 50mM, 80mM, 100mM), the MOPSO buffer has a pH of 6.2-7.6 (e.g., 6.2, 6.5, 6.7, 7.0, 7.3, 7.6), guanidinium isothiocyanate has a concentration of 1-10M (e.g., 1M, 2M, 3M, 5M, 8M, 10M), the nonionic detergent has a concentration of 0.1-5% (e.g., 0.1%, 0.5%, 1.0%, 1.5%, 2%, 5%) the anionic detergent has a concentration of 0.01-1% (e.01%, 0.05%, 0.10%, 0.50%, 1%) the dithiothreitol has a concentration of 1-20M (e.g., 1M, 2M, 3M, 5M, 10M, 20M), the EDTA has a concentration of 0.1-200mM (e.1%, 1.1%, 1.1.5M), 0.5mM, 1mM, 10mM, 50mM, 200mM), isopropanol at a concentration of 10% to 99.5% (e.g., 10%, 20%, 50%, 80%, 99.5%) by mass, sodium chloride at a concentration of 1 to 200mM (e.g., 1mM, 10mM, 30mM, 50mM, 100mM, 200mM) by mass, and a preservative at a concentration of 0.01% to 0.1% (e.g., 0.01%, 0.05%, 0.10% by mass).
In one embodiment, the concentration of sodium chloride is 1-200mM (e.g., 1mM, 10mM, 50mM, 200mM) and the concentration of ethanol is 10% -100% (e.g., 10%, 20%, 50%, 80%, 100%) by mass in the rinse buffer.
In one embodiment, in the magnetic bead adsorption solution, the concentration of 0.5-1.5 μm magnetic beads is 10-100mg/mL (e.g., 10mg/mL, 20mg/mL, 50mg/mL, 80mg/mL, 100mg/mL), the concentration of 40-300nm magnetic beads is 10-100mg/mL (e.g., 10mg/mL, 20mg/mL, 50mg/mL, 80mg/mL, 100mg/mL), the concentration of MOPSO buffer is 10-100mM (e.g., 1mM, 10mM, 50mM, 100mM), and the concentration of preservative is 0.01-0.1% (e.g., 0.01%, 0.05%, 0.10%) by mass.
In one embodiment, the ratio of 0.5-1.5 μm magnetic beads to 40-300nm magnetic beads is 0.5-3:1 (e.g., 0.5:1, 1:1, 1.5:1, 2:1, 3: 1).
In one embodiment, the preservative may be ProClin 300.
In one embodiment, the 0.5-1.5 μm magnetic beads are: silicon-based magnetic beads or silicon hydroxyl magnetic beads with the particle size of 0.5-1.5 mu m (0.5 mu m, 1.5 mu m). The 40-300nm magnetic beads are: silicon-based magnetic beads or silicon hydroxyl magnetic beads with the particle size of 40-300 nm.
In the examples, M represents the concentration unit mol per liter (mol/L) and mM represents the concentration unit millimole per liter (mmol/L).
The preparation method of the MOPSO buffer solution comprises the following steps:
(1) prepared concentration 50mM solution (A): weighing MOPSO11.263g, and using deionized water to fix the volume to 1L;
(2) prepared solution (B) with concentration of 50 mM: weighing NaOH2g, and fixing the volume to 1L by using deionized water;
(3) the MOPSO buffer at pH3.9 was: solution (a) solution (B) 5: 0;
the MOPSO buffer at pH6.2 was: solution (a) solution (B) 5: 1;
the MOPSO buffer at pH6.6 was: solution (A) solution (B) 5: 2;
the MOPSO buffer at pH7.0 was: solution (a) solution (B) 5: 3;
the MOPSO buffer at pH7.4 was: solution (a) solution (B) 5: 4.
The method for extracting nucleic acid of the application is realized based on the nucleic acid extraction kit of the embodiment, and comprises the following steps:
s10: and mixing the lysis buffer solution, the proteinase K freeze-dried powder and the magnetic bead adsorption solution with the sample so as to obtain the magnetic beads with adsorbed nucleic acids.
Specifically, according to the parts by volume, 10-30 parts of a sample, 30-50 parts of a lysis buffer solution, 1-50 parts of proteinase K freeze-dried powder and 1-5 parts of a magnetic bead adsorption solution are placed in a centrifuge tube, and are uniformly mixed in a vortex mode to obtain a first mixture. The first mixture was incubated at room temperature with shaking.
S20: and washing the magnetic beads adsorbed with the nucleic acid by using a rinsing buffer solution, carrying out solid-liquid separation after washing, and collecting the magnetic beads adsorbed with the nucleic acid.
Specifically, after the incubation is finished, the centrifuge tube is placed on a magnetic frame, standing is carried out until all the magnetic beads with the nucleic acid adsorbed thereon are adsorbed on the tube wall, and the supernatant is removed. Removing the magnetic frame, and adding 50-100 parts of rinsing buffer solution into the centrifuge tube to fully clean the magnetic beads adsorbed with the nucleic acid; and then placing the centrifuge tube on a magnetic frame, standing until all the magnetic beads with the nucleic acid adsorbed are adsorbed to the tube wall, and removing the supernatant.
S30: and (3) eluting the washed magnetic beads adsorbed with the nucleic acid by using an eluent, carrying out solid-liquid separation after elution, and collecting supernatant so as to obtain a nucleic acid solution.
Specifically, 3-20 parts of eluent is added into the centrifugal tube, the centrifugal tube is vibrated for 5-10 minutes at 50-70 ℃, and the centrifugal tube is shaken for 10-15 times every 1 minute so that the magnetic beads in the centrifugal tube are always uniformly distributed; after the incubation is finished, the centrifugal tube is vortexed at high speed for 10-15 times; and (3) placing the centrifugal tube on a magnetic frame, standing until all the magnetic beads are adsorbed to the tube wall, and sucking supernatant into a ribozyme-free clean centrifugal tube to obtain the purified nucleic acid solution.
To better illustrate the objects, technical solutions and advantages of the present application, the present application will be further described with reference to specific embodiments.
Example 1
In one embodiment of the nucleic acid extraction kit of the present application, the nucleic acid extraction kit includes lysis buffer, proteinase K lyophilized powder, rinsing buffer, eluent and magnetic bead adsorption solution.
Wherein the proteinase K freeze-dried powder is obtained by freeze-drying a proteinase K solution. Specifically, 200 μ L of proteinase K solution is transferred to a freeze-drying bottle, the freeze-drying bottle is placed in a low-temperature ethanol bath below-20 ℃ and slightly rotated to ensure that the solute is uniformly coated on the wall of the bottle as thin as possible, and after the solution is evaporated to dryness, the freeze-drying bottle is firmly fixed on a freeze-drying machine for 10 hours; taking down the freeze-drying bottle with the freeze-drying powder on the inner wall, vacuumizing, sealing, and storing at 4-30 deg.C.
The specific components of the lysis buffer, proteinase K solution, rinse buffer, eluent and magnetic bead adsorption solution are shown in table 1.
TABLE 1 buffer composition table
The preparation process of each reagent in the nucleic acid extraction kit is as follows:
(1) lysis buffer: weighing the components according to the table 1, dissolving the components in deionized water, and adjusting the pH to 7.3 by using a 50mM NaOH solution;
(2) protease K freeze-dried powder: weighing the components according to the table 1, dissolving in deionized water, taking 20 mu L, subpackaging into freeze-drying bottles, and freeze-drying to obtain 1 part of proteinase K freeze-dried powder;
(3) rinsing buffer: weighing the components according to the table 1, and mixing uniformly to obtain the product.
(4) Eluent: DEPC (diethylpyrocarbonate) was added to deionized water to a final concentration of 0.05% (V/V), and after standing at room temperature (22-25 ℃) for 10-12 hours, DEPC was removed at 121 ℃ for 20 minutes under high pressure and left at room temperature for further use.
(5) Magnetic bead adsorption solution: preparing 50mM MOPSO buffer solution with pH of 7.3, adding 1um magnetic bead, 200nm magnetic bead and antiseptic shown in Table 1, and mixing.
Example 2
The method for extracting nucleic acid comprises the following steps:
(1) 200 mu L of sample, 500 mu L of lysis buffer prepared in example 1, 1 part of proteinase K freeze-dried powder prepared in example 1 and 10 mu L of magnetic bead adsorption solution prepared in example 1 are weighed and placed in a centrifuge tube, and are uniformly mixed by vortex to obtain a first mixture. The first mixture was incubated at room temperature with shaking.
(2) After incubation, placing the centrifuge tube in a magnetic frame, standing until all the magnetic beads with the adsorbed nucleic acid are adsorbed to the tube wall, and removing supernatant. And removing the magnetic frame, adding 700 mu L of the rinsing buffer solution prepared in the example 1 into the centrifuge tube to fully clean the magnetic beads adsorbed with the nucleic acid, then placing the centrifuge tube on the magnetic frame, standing until all the magnetic beads adsorbed with the nucleic acid are adsorbed to the tube wall, and removing the supernatant.
(3) Adding 100 mu L of the eluent prepared in the embodiment 1 into a centrifugal tube, shaking the centrifugal tube for 5-10 minutes at 56 ℃, and shaking the centrifugal tube for 10-15 times every 1 minute so as to keep the magnetic beads in the centrifugal tube in a uniformly distributed state all the time; after the incubation is finished, the centrifugal tube is vortexed at high speed for 10-15 times; and (3) placing the centrifugal tube on a magnetic frame, standing until all the magnetic beads are adsorbed to the tube wall, and sucking supernatant into a ribozyme-free clean centrifugal tube to obtain the purified nucleic acid solution.
Example 3
Performance detection
(1) Determination of extraction yield
The method of example 2 was used to extract nucleic acids from serum samples, and spectrophotometric detection was used to determine the extraction yield, which was found to be equal to or greater than 10 ng/. mu.L, based on the absorbance of the solution at 260 nm. The results of the experiment are shown in table 2.
(2) Determination of nucleic acid purity
Nucleic acids in serum samples were extracted using the method of example 2, optical density values (OD) at 260nm and 280nm were measured using a UV spectrophotometer, and OD260/280 was calculated, with OD260/280 being between 1.6 and 2.0 as passing. The results of the experiment are shown in table 2.
(3) Determination of extraction efficiency
The HBVDNA standard substance and the HCVRNA virus national standard substance and the human genome DNA standard substance (both prepared by using the negative plasma dilution standard substance) with the concentration of 25ug/ml are extracted by using the method of example 2, and the concentration ratio before and after extraction is calculated, so that the result is that the HBVDNA standard substance, the HCVRNA virus national standard substance and the human genome DNA standard substance are qualified when the concentration ratio is more than or equal to 85 percent. The results of the experiment are shown in table 2.
(4) Precision in batch
Repeating the extraction for 10 times for 50ng/ml national standard substance, and calculating the imprecision degree in the batch
The CV value of 10% or less was found to be acceptable. The results of the experiment are shown in table 2.
TABLE 2 detection of the Performance of nucleic acid extracts
Example 4
Stability test
The performance of (1) to (4) in example 3 was tested again after subjecting the extraction products of example 3 to accelerated denaturation at 37 ℃ for 7 days. The results of the experiment are shown in table 3.
TABLE 3 detection of the Performance of the nucleic acid extracts after 7 days of accelerated denaturation at 37 ℃
In conclusion, the nucleic acid extraction method based on the nucleic acid extraction kit is adopted to extract the nucleic acid from the sample, and the nucleic acid extraction product is identified, so that the extraction yield, the nucleic acid purity, the extraction efficiency and the batch precision of the nucleic acid extraction product are all qualified, and the extraction yield, the nucleic acid purity, the extraction efficiency and the batch precision of the nucleic acid extraction product after being subjected to accelerated denaturation at 37 ℃ for 7 days are all qualified.
Different from the condition of the prior art, the nucleic acid extraction kit comprises a lysis buffer solution, proteinase K freeze-dried powder, a rinsing buffer solution, an eluent and a magnetic bead adsorption solution, wherein the proteinase K freeze-dried powder can be stably stored at room temperature for a long time, so that the stability of the preparation is improved, and the defect that the proteinase K activity is obviously reduced due to frequent opening of the kit when the proteinase K solution is stored at-20 ℃ in a freezing way in the prior art is overcome. When the protease K freeze-dried powder is used, the protease K freeze-dried powder is dissolved in a lysis buffer solution and a magnetic bead adsorption solution, and the operation is convenient.
In addition, guanidine isothiocyanate is used together with nonionic detergent and anionic detergent, so that the aim of quickly cracking cells at room temperature can be fulfilled. Meanwhile, only one-step rinsing is needed due to the reduction of the salt ion concentration.
The above embodiments are merely examples and are not intended to limit the scope of the present disclosure, and all modifications, equivalents, and flow charts using the contents of the specification and drawings of the present disclosure or those directly or indirectly applied to other related technical fields are intended to be included in the scope of the present disclosure.
Claims (10)
1. A nucleic acid extraction kit, comprising: lysis buffer solution, proteinase K freeze-dried powder, rinsing buffer solution, eluent and magnetic bead adsorption solution;
the proteinase K freeze-dried powder comprises: MOPSO, proteinase K, a preservative and a stabilizer.
2. The kit according to claim 1, wherein the storage temperature of the nucleic acid extraction kit is 4 to 30 ℃.
3. The kit according to claim 1,
the lysis buffer comprises: MOPSO buffer, guanidinium isothiocyanate, nonionic detergent, anionic detergent, EDTA, dithiothreitol, isopropanol, sodium chloride, and preservative;
the rinse buffer comprises: sodium chloride and ethanol;
the eluent comprises: rnase-free water;
the magnetic bead adsorption solution comprises: 0.5-1.5 μm magnetic bead, 40-300nm magnetic bead, MOPSO buffer solution, and antiseptic.
4. The kit according to claim 3,
in the lysis buffer solution, the concentration of the MOPSO buffer solution is 10-100mM, the pH value of the MOPSO buffer solution is 6.2-7.6, the mass percent concentration of guanidine isothiocyanate is 0.1-5%, the mass percent concentration of non-ionic detergent is 0.1-5%, the mass percent concentration of anionic detergent is 0.01-1%, the concentration of dithiothreitol is 1-20M, the mass percent concentration of EDTA is 0.1-200mM, the mass percent concentration of isopropanol is 10-99.5%, the concentration of sodium chloride is 1-200mM, and the mass percent concentration of preservative is 0.01-0.1%.
5. The kit according to claim 3,
in the rinsing buffer solution, the concentration of the sodium chloride is 1-200mM, and the concentration of the ethanol is 10% -100%.
6. The kit according to claim 3,
in the magnetic bead adsorption solution, the concentration of 0.5-1.5 μm magnetic beads is 10-100mg/mL, the concentration of 40-300nm magnetic beads is 10-100mg/mL, the concentration of MOPSO buffer solution is 10-100mM, and the mass percentage concentration of the preservative is 0.01-0.1%.
7. The kit according to claim 3,
the magnetic beads with the diameter of 0.5-1.5 μm: the ratio of the 40-300nm magnetic beads is 0.5-3: 1.
8. the kit according to claim 3,
the nonionic detergent is ethyl phenyl polyethylene glycol;
the anionic detergent is sodium dodecyl sulfate;
the preservative is ProClin 300;
the stabilizer is dextran and mannitol.
The magnetic beads with the diameter of 0.5-1.5 μm are: silicon-based magnetic beads or silicon hydroxyl magnetic beads with the particle size of 0.5-1.5 mu m;
the 40-300nm magnetic beads are: silicon-based magnetic beads or silicon hydroxyl magnetic beads with the particle size of 40-300 nm.
9. A method for extracting nucleic acid, which is based on the nucleic acid extraction kit according to any one of claims 1 to 8, comprising the steps of:
mixing the lysis buffer solution, the proteinase K freeze-dried powder and the magnetic bead adsorption solution with a sample so as to obtain magnetic beads with adsorbed nucleic acids;
washing the magnetic beads adsorbed with the nucleic acid by using a rinsing buffer solution, carrying out solid-liquid separation after washing, and collecting the magnetic beads adsorbed with the nucleic acid;
and eluting the washed magnetic beads adsorbed with the nucleic acid by using an eluent, carrying out solid-liquid separation after elution, and collecting supernatant so as to obtain a nucleic acid solution.
10. The method of claim 9,
according to the volume parts, the lysis buffer solution is 30-50 parts, the proteinase K freeze-dried powder is 0.1-50 parts, the magnetic bead adsorption solution is 1-5 parts, the sample is 10-30 parts, the rinsing buffer solution is 50-100 parts, and the eluent is 3-20 parts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011480855.0A CN114634925A (en) | 2020-12-15 | 2020-12-15 | Nucleic acid extraction kit and method for extracting nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011480855.0A CN114634925A (en) | 2020-12-15 | 2020-12-15 | Nucleic acid extraction kit and method for extracting nucleic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114634925A true CN114634925A (en) | 2022-06-17 |
Family
ID=81944842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011480855.0A Pending CN114634925A (en) | 2020-12-15 | 2020-12-15 | Nucleic acid extraction kit and method for extracting nucleic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114634925A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115074356A (en) * | 2022-08-18 | 2022-09-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | New coronavirus nucleic acid extraction promoter and application thereof |
| CN116042792A (en) * | 2022-09-05 | 2023-05-02 | 杭州拜赛思生物科技有限责任公司 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005072456A2 (en) * | 2004-01-28 | 2005-08-11 | Gerard Biotech, Llc | Single use lyophilized enzymatic reagents, and kits and methods for using same |
| CN107177572A (en) * | 2017-06-19 | 2017-09-19 | 广州和实生物技术有限公司 | A kind of lyophilized technique of Proteinase K |
| CN108330127A (en) * | 2018-03-09 | 2018-07-27 | 佛山市优特医疗科技有限公司 | A kind of magnetic bead mixed liquor, nucleic acid preservation method, nucleic acid extracting reagent and kit |
-
2020
- 2020-12-15 CN CN202011480855.0A patent/CN114634925A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005072456A2 (en) * | 2004-01-28 | 2005-08-11 | Gerard Biotech, Llc | Single use lyophilized enzymatic reagents, and kits and methods for using same |
| CN107177572A (en) * | 2017-06-19 | 2017-09-19 | 广州和实生物技术有限公司 | A kind of lyophilized technique of Proteinase K |
| CN108330127A (en) * | 2018-03-09 | 2018-07-27 | 佛山市优特医疗科技有限公司 | A kind of magnetic bead mixed liquor, nucleic acid preservation method, nucleic acid extracting reagent and kit |
Non-Patent Citations (3)
| Title |
|---|
| 张建中等: "应用于即时检测的核酸提取与扩增试剂冷冻干燥研究", 《中国知网硕士电子期刊》, no. 9, pages 32 * |
| 曹墨菊: "《植物生物技术概论》", vol. 1, 中国农业大学出版社, pages: 133 * |
| 苏燕等: "《医学生物化学与分子生物学实验技术双语教程》", vol. 2, 30 September 2015, 中国农业大学出版社, pages: 111 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115074356A (en) * | 2022-08-18 | 2022-09-20 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | New coronavirus nucleic acid extraction promoter and application thereof |
| CN116042792A (en) * | 2022-09-05 | 2023-05-02 | 杭州拜赛思生物科技有限责任公司 | Telomerase activity gene detection kit, primer set, probe primer and detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1618194B1 (en) | Compositions and methods for using a solid support to purify rna | |
| CA2790941C (en) | Method for isolating rna from a rna and dna containing sample | |
| US7303876B2 (en) | Compositions, methods, and kits for isolating nucleic acids using surfactants and proteases | |
| JP5588097B2 (en) | Methods for nucleic acid isolation | |
| CN101124321B (en) | Compositions and methods for purifying nucleic acids from stabilization reagents | |
| CN111088249A (en) | Use method of metagenome sample de-hosting extraction kit | |
| WO2006026248A1 (en) | Compositions and methods employing zwitterionic detergent combinations | |
| EP1694692B1 (en) | Formulations and methods for denaturing proteins | |
| AU759401B2 (en) | Improved method for the isolation of nucleic acid | |
| CN114634925A (en) | Nucleic acid extraction kit and method for extracting nucleic acid | |
| JP2013539366A (en) | Method for isolating target nucleic acids, including small target nucleic acids, in high yield | |
| CN110129314B (en) | Amino acid buffer solution and plasma free DNA extraction kit | |
| US20230257733A1 (en) | Method for isolating nucleic acid | |
| TWI407994B (en) | Method, agent, and kit for isolating nucleic acids | |
| EP4163371A1 (en) | Nucleic acid extraction method and application | |
| KR100454869B1 (en) | Cell lysis buffer for extracting DNA and extraction method by using thereof | |
| WO2008035991A2 (en) | A nucleic acid extraction method | |
| CN113355320A (en) | Lysis, binding, washing and/or elution reagents for separating and/or purifying nucleic acids | |
| CN116334071A (en) | Nucleic acid extraction kit for detecting pathogenic microorganisms and extraction method thereof | |
| CN113005117B (en) | Magnetic bead drying protection solution, drying magnetic bead and preparation method thereof | |
| EP3619309B1 (en) | Rapid purification of high quality nucleic acids from biological samples | |
| AU2004233035B2 (en) | Compositions and methods for using a solid support to purify RNA | |
| CN117230052A (en) | All-purpose nuclease stability protective agent, liquid enzyme preparation and preparation method thereof | |
| CN115011588A (en) | Bacterial genome DNA extraction kit and use method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |