CN117511932A - Kit for extracting or purifying nucleic acid without proteinase K - Google Patents
Kit for extracting or purifying nucleic acid without proteinase K Download PDFInfo
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- CN117511932A CN117511932A CN202311758256.4A CN202311758256A CN117511932A CN 117511932 A CN117511932 A CN 117511932A CN 202311758256 A CN202311758256 A CN 202311758256A CN 117511932 A CN117511932 A CN 117511932A
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 61
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 61
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 61
- 108010067770 Endopeptidase K Proteins 0.000 title claims abstract description 26
- 238000000605 extraction Methods 0.000 claims abstract description 74
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 44
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 28
- 239000006166 lysate Substances 0.000 claims abstract description 24
- 238000000746 purification Methods 0.000 claims abstract description 21
- 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 18
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 14
- 239000001632 sodium acetate Substances 0.000 claims abstract description 14
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 14
- 239000011780 sodium chloride Substances 0.000 claims abstract description 14
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000004202 carbamide Substances 0.000 claims abstract description 9
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108010077895 Sarcosine Proteins 0.000 claims abstract description 6
- 229940048098 sodium sarcosinate Drugs 0.000 claims abstract description 6
- 108700004121 sarkosyl Proteins 0.000 claims description 36
- BACYUWVYYTXETD-UHFFFAOYSA-N N-Lauroylsarcosine Chemical compound CCCCCCCCCCCC(=O)N(C)CC(O)=O BACYUWVYYTXETD-UHFFFAOYSA-N 0.000 claims description 34
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 28
- 229910052708 sodium Inorganic materials 0.000 claims description 28
- 239000011734 sodium Substances 0.000 claims description 28
- 239000011324 bead Substances 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- 208000007407 African swine fever Diseases 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 239000001509 sodium citrate Substances 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 3
- 229910052710 silicon Inorganic materials 0.000 claims description 3
- 239000010703 silicon Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 108091005804 Peptidases Proteins 0.000 claims 9
- 102000035195 Peptidases Human genes 0.000 claims 9
- 235000019833 protease Nutrition 0.000 claims 9
- 238000001514 detection method Methods 0.000 abstract description 16
- 230000009089 cytolysis Effects 0.000 abstract description 8
- 238000000034 method Methods 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 10
- 238000012795 verification Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 241000701386 African swine fever virus Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 241001493065 dsRNA viruses Species 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 2
- 102000011931 Nucleoproteins Human genes 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- YFVGRULMIQXYNE-UHFFFAOYSA-M lithium;dodecyl sulfate Chemical compound [Li+].CCCCCCCCCCCCOS([O-])(=O)=O YFVGRULMIQXYNE-UHFFFAOYSA-M 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- XMTQQYYKAHVGBJ-UHFFFAOYSA-N 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA Chemical compound CN(C)C(=O)NC1=CC=C(Cl)C(Cl)=C1 XMTQQYYKAHVGBJ-UHFFFAOYSA-N 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 239000005510 Diuron Substances 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 229920001284 acidic polysaccharide Polymers 0.000 description 1
- 150000004805 acidic polysaccharides Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- -1 aromatic amino acids Chemical class 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 125000000400 lauroyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229940125956 metalloenzyme inhibitor Drugs 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 229940016590 sarkosyl Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229920001187 thermosetting polymer Polymers 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000003612 virological effect Effects 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 relates to the technical field of molecular biology detection, in particular to a kit for extracting or purifying nucleic acid without proteinase K, which comprises a lysate, wherein the lysate comprises 1.0-5.0 mol/L guanidine isothiocyanate, 10-50 mmol/L sodium acetate, 10-30 mmol/L EDTA, 0.5-5.0 mol/L NaCl, 0.1-3% of urea, 10-70% of isopropanol by volume, CTAB and N-lauroyl sodium sarcosinate, and the mass concentration ratio of the CTAB to N-lauroyl sodium sarcosinate is 10-50. The nucleic acid extraction or purification kit provided by the invention has the advantages that the components and the concentration of the lysate are properly adjusted, the obtained lysis system has strong lysis capacity and high lysis efficiency, and the extraction efficiency is equivalent to that of the kit containing proteinase K under the condition of not containing proteinase K.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a kit for extracting or purifying nucleic acid without protease K.
Background
From African swine fever, which is an animal virulent infectious disease, to a novel coronavirus, a huge economic loss is caused to the pig industry; the latter is a new human infectious disease, which constitutes a great threat to human health and life, resulting in a great socioeconomic loss. Therefore, rapid and accurate pathogen diagnosis is an important premise for effective treatment, disease monitoring and disease spread control. With the development of detection technology, pathogenic nucleic acid detection has become the mainstream detection means due to its high sensitivity and high specificity detection performance.
Nucleic acid extraction is the first step of pathogen nucleic acid detection, and the most commonly used nucleic acid extraction methods at present are a magnetic bead adsorption method and a silica gel membrane adsorption column method. The silica gel membrane adsorption column method is rapid and simple, the purity of the obtained nucleic acid is high, but the extraction efficiency of small fragment DNA or RNA is not high, and the method is not suitable for an automatic extraction process. The magnetic bead method uses superparamagnetic nano microsphere as carrier, can adsorb nucleic acid, can quickly separate and purify DNA or RNA, and can be implemented in the form of automatic working platform without need of centrifugation or filtration in the process of extraction, and its extraction flux is high and automation degree is good.
Proteinase K, a serine protease with a broader cleavage activity, is able to cleave the carboxyl-terminal peptide bond of aliphatic and aromatic amino acids. Since proteinase K is stable in urea and SDS and also has the ability to degrade natural proteins, viral coat proteins can be cleaved in viral nucleic acid extraction, exposing the nucleic acids; degrading histone closely combined with nucleic acid to make the nucleic acid free to the extracting solution, which is favorable for further purification; meanwhile, proteinase K can degrade RNase to prevent degradation of viral RNA, which is beneficial to nucleic acid detection. However, in the nucleic acid extraction process, proteinase K is added, which increases the operation steps, the extraction time and the reagent cost, and is time-consuming and labor-consuming for extracting nucleic acid with a large sample size. In addition, proteinase K is not thoroughly washed during the extraction process and can inhibit downstream PCR.
The existing magnetic bead method still has the problem of long extraction time, the whole extraction process generally needs about 30 minutes, and when dealing with emergency, the method can not meet the requirement of simpler and faster nucleic acid extraction under a large number of samples. Therefore, there is a need for improved magnetic bead nucleic acid extraction systems, and development of a set of simpler, faster, suitable for automated work platform nucleic acid extraction and purification reagents, for providing high quality nucleic acid templates for pathogenic nucleic acid detection, and improving detection efficiency and detection accuracy.
Disclosure of Invention
The invention aims to provide a kit for extracting or purifying proteinase K nucleic acid, which solves the technical problems that proteinase K is not thoroughly cleaned and inhibits downstream PCR reaction in the prior art.
The invention discloses a kit for extracting or purifying nucleic acid without protease K, which comprises a lysate, wherein the lysate comprises 1.0-5.0 mol/L guanidine isothiocyanate, 10-50 mmol/L sodium acetate, 10-30 mmol/L EDTA, 0.5-5.0 mol/L NaCl, 0.1-3% of urea, 10-70% of isopropanol by volume, CTAB and N-lauroyl sarcosine sodium, and the mass concentration ratio of the CTAB and N-lauroyl sarcosine sodium substances is 10-50.
Further, the concentration of CTAB is 10 mmol/L-30 mmol/L, and the concentration of N-lauroyl sarcosinate is 0.5 mmol/L-2 mmol/L;
preferably, the concentration of CTAB and sodium N-lauroyl sarcosinate is 20mM and 1.5mM, respectively, for the new crown swab sample; for oronasal swab samples, the concentration of CTAB and N-lauroyl sarcosinate was 15mM and 1.0mM, respectively.
Preferably, for a new crown swab sample, the lysate comprises 3mol/L guanidine isothiocyanate, 30mmol/L sodium acetate, 10mmol/L EDTA, 3mol/L NaCl, 2% urea by mass and 20% isopropanol by volume; for an African swine fever sample, the lysate comprises 3.5mol/L guanidine isothiocyanate, 30mmol/L sodium acetate, 10mmol/L EDTA, 3mol/L NaCl, 2% urea by mass and 20% isopropanol by volume;
further, the kit also comprises a washing liquid 1, wherein the washing liquid 1 comprises 10-50 mmol/L of sodium acetate, 5-30 mmol/L of EDTA, 0.5-3.0 mol/L of NaCl and 30-70% of isopropanol or absolute ethyl alcohol in volume ratio;
preferably, the washing liquid 1 comprises 15mmol/L sodium acetate, 20mmol/L EDTA,0.8mol/L NaCl and 50% isopropyl alcohol by volume;
further, the kit also comprises a washing liquid 2, wherein the washing liquid 2 comprises 0.5-3 mmol/L of sodium citrate, 1-10 mmol/L of EDTA, and 40-90% of isopropanol or absolute ethyl alcohol by volume ratio; the trisodium citrate in the washing liquid can control the ionic strength of a reaction system, and has a certain buffer function, so that the stability of nucleic acid is facilitated, and the damage to the nucleic acid is reduced.
Preferably, the washing liquid 2 comprises 2.0mmol/L sodium citrate, 1.0mmol/L EDTA and 60% absolute ethyl alcohol by volume;
further, the kit also comprises an eluent, wherein the eluent comprises 0.1 mmol/L-10 mmol/L Tris;
preferably, the concentration of Tris in the eluent is 0.1mmol/L;
further, the kit also comprises a magnetic bead mixed solution, wherein the magnetic bead mixed solution comprises magnetic beads and a magnetic bead diluent, the functional groups carried by the magnetic beads comprise but are not limited to amino groups, hydroxyl groups, carboxyl groups or silicon hydroxyl groups, and the concentration of the magnetic beads in the magnetic bead mixed solution is 15mg/ml;
further, the magnetic bead diluent comprises 0.1-2 mol/L sodium chloride, 0.1-5 mmol/L EDTA and 30-90% by volume of isopropanol.
Preferably, the functional group in the magnetic bead mixture is a silicon hydroxyl group.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the nucleic acid extraction or purification kit, the components and the concentration of the lysate are properly adjusted, so that the obtained lysis system has strong lysis capacity and high lysis efficiency, and the extraction efficiency is equivalent to that of the kit containing proteinase K under the condition of not containing proteinase K;
2. the nucleic acid extraction or purification kit simplifies the process of nucleic acid extraction and purification, reduces the time required by one detection sample, and is beneficial to clinical detection of a large number of samples;
3. the nucleic acid extraction or purification kit provided by the invention has the advantages that the extraction system does not contain proteinase K, so that the influence of downstream PCR reaction caused by incomplete cleaning of proteinase K is solved, and the extraction efficiency is improved.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments.
The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid, or analogue units thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.
The nucleic acid extraction or purification kit of the invention improves the lysate in the kit, and discovers that N-lauroyl sarcosine is an anionic surfactant in the lysate. It has amphipathy, and has both hydrophobic 14 carbon chain (lauroyl) and hydrophilic carboxyl. Sarkosyl can penetrate cells and lyse lipids and proteins; can decompose the fat membrane of the cells and release the cell content; in addition, the initiation of DNA transcription can be suppressed. CTAB (cetyltrimethylammonium bromide, hexadecyl trimethyl ammonium Bromide), which is a cationic detergent, can dissolve cell membranes and form a complex with nucleic acid, has the characteristic of precipitating nucleic acid and acidic polysaccharide from a low-ionic strength solution, and in a high-ionic strength solution (> 0.7mol/L NaCl), CTAB forms a complex with protein and polysaccharide, so that the cleavage efficiency is improved, and the cleavage effect is enhanced; when the concentration ratio of N-lauroyl sarcosine sodium and CTAB with coordinated functions in the lysate is proper, proteinase K is not needed to be added in the lysis process, so that DNA can be selectively kept in the solution, and the lysis time can be shortened.
Based on the extraction or purification kit, the concentration of each substance in the lysate is properly adjusted, and particularly the concentration proportion of CTAB and N-lauroyl sarcosine sodium with synergistic effect in the system is explored, so that the system finally achieves the optimal synergistic effect, the cleavage time is shortened under the condition that proteinase K is not required to be added, and the extraction efficiency is superior to that of a control reagent (containing proteinase K and the extraction time is long).
The kit of the invention also contains guanidine isothiocyanate, which is used as a powerful protein denaturant, can rapidly dissolve protein, causes the cell structure to be broken, and can destroy the secondary structure of nucleoprotein so as to rapidly separate the nucleoprotein from nucleic acid; EDTA as a chelating divalent metalloenzyme inhibitor, chelating Mg 2+ Or Mn of 2+ Ions, which inhibit the activity of DNase in cells and can inhibit the degradation of DNA by deoxyribonuclease (DNase); sodium acetate is mainly used for promoting DNA precipitation and is aggregated into insoluble network polymers, and high concentration of sodium acetate can cause precipitation of protein-SDS complex and RNA molecules with high molecular weight; isopropanol mainly acts to precipitate or precipitate DNA; the magnetic beads are the media for nucleic acid transfer adsorption.
The use method of the kit of the invention is that the Tianlong GeneRotex96 extractor or other instruments of the same type are used for automatic extraction, and the method comprises the following steps:
automated extraction using a diuron GeneRotex96 extractor or other equivalent type of instrument, comprises the steps of:
1. according to the number of extracted samples, 400. Mu.L of sample and 450. Mu.L of lysate were sequentially added in column 1/7 of the 96-well plate.
2. Adding 300 mu L of magnetic bead mixed solution into the 2/8 th column of a 96-deep well plate according to the number of extracted samples;
3. adding 700 mu L of washing liquid into the 3/9 th column of the 96-deep well plate according to the number of the extracted samples;
4. adding 800 mu L of washing liquid into the 4/10 th column of the 96-deep well plate according to the number of the extracted samples;
5. adding 80 mu L of eluent into the 6/12 th column of the 96 deep-hole plate according to the number of extracted samples;
6. before adding the magnetic bead mixed solution, fully and uniformly mixing the magnetic beads, if the number of the extracted samples is large, the magnetic beads are recommended to be resuspended once every 8 sample holes are added;
7. extraction was performed using a Tianlong GeneRotex96 automatic extractor, with the extraction procedure shown in Table 1, for about 8 minutes.
TABLE 1 extraction program of the invention for matching GeneRotex96 automatic extractor
After the end of the automated procedure, column 6/12 (note the effective working well) of the eluate was transferred to a clean nuclease-free centrifuge tube.
The extraction effect of the nucleic acid extraction or purification kit of the present invention is verified as described in the following examples. Wherein, in each example, the concentration unit mmol/L of the component is abbreviated as mM, and the mol/L is abbreviated as M.
EXAMPLE 1 lysate of the present invention
The invention explores the concentration and proportion range of CTAB and N-lauroyl sodium sarcosinate in the lysate, when the concentration of CTAB or N-lauroyl sodium sarcosinate is too high, on one hand, the binding efficiency of nucleic acid and magnetic beads is unbalanced, so that the extraction efficiency is reduced, and on the other hand, the downstream PCR amplification is inhibited; when the concentration of CTAB and N-lauroyl sarcosine sodium is too low, the cracking efficiency of the lysate is reduced, and the extraction efficiency is also reduced. Thus, this example demonstrates the concentration ranges of both CTAB and N-lauroyl sarcosine sodium.
In this example, the concentration of guanidine isothiocyanate in the lysate of the present invention was 3M, the concentration of sodium acetate was 30mM, the concentration of EDTA was 10mM, the concentration of NaCl was 3M, the mass percentage of urea was 2%, and the volume ratio of isopropyl alcohol was 20%.
Verification method and result: the ASFV negative swab sample is used for diluting the ASFV sample swab sample to about 36CT, CTAB and N-lauroyl sarcosine sodium with the following concentrations are used for extraction, the same sample is used for each experimental group, each experimental group is respectively extracted for 20 times, the detection rate is calculated, and the detection rate is required to be more than or equal to 95%. The detailed results are shown in tables 2 to 7.
TABLE 2 concentration Range sensitivity verification results for both CTAB and N-lauroyl sarcosine sodium (CTAB concentration 5mM, N-lauroyl sarcosine sodium concentration 0.1-3 mM)
TABLE 3 concentration Range sensitivity verification results for both CTAB and N-lauroyl sarcosine sodium (CTAB concentration 10mM, N-lauroyl sarcosine sodium concentration 0.1-3 mM)
TABLE 4 concentration Range sensitivity verification results for both CTAB and N-lauroyl sarcosine sodium (CTAB concentration 15mM, N-lauroyl sarcosine sodium concentration 0.1-3 mM)
TABLE 5 concentration Range sensitivity verification results for both CTAB and N-lauroyl sarcosine sodium (CTAB concentration 20mM, N-lauroyl sarcosine sodium concentration 0.1-3 mM)
TABLE 6 concentration Range sensitivity verification results for both CTAB and N-lauroyl sarcosine sodium (CTAB concentration 30mM, N-lauroyl sarcosine sodium concentration 0.1-3 mM)
TABLE 7 concentration Range sensitivity verification results for both CTAB and N-lauroyl sarcosine sodium (CTAB concentration 40mM, N-lauroyl sarcosine sodium concentration 0.1-3 mM)
From the data in tables 2 to 7, it was found that when the concentration of CTAB was 10 to 30mM and the concentration of N-lauroyl sarcosine sodium was 0.5 to 2.0mM, the detection rate of the kit containing the lysate of the present invention was in accordance with the standard, and at this time, the concentration ratio of CTAB to N-lauroyl sarcosine sodium in the kit of the present invention was 5 to 60, and after the ratio was determined, the precision test was conducted on the ratio range, and the system stability was determined, so that the concentration ratio of CTAB to N-lauroyl sarcosine sodium was further verified by CT value.
Verification method and result: the ASFV negative swab sample is used for diluting the ASFV sample swab sample to medium concentration, CTAB and N-lauroyl sarcosine sodium with the concentration ratio below are used for extraction, the same sample is used for each experimental group, each experimental group is respectively extracted for 4 times, CV value of CT value is calculated, and CV value of CT value is less than or equal to 2%. The detailed results are shown in Table 8.
TABLE 8 results of accuracy validation of the ratio Range of CTAB to sodium N-lauroyl sarcosinate
According to the standard requirement that CV is less than or equal to 2%, when the proportion of CTAB and N-lauroyl sarcosine sodium in the lysate is within the range of 10-50, the performance of the kit using the lysate meets the extraction requirement.
EXAMPLE 2 kits of the invention
The components of the kit system of the invention are shown in Table 9 for the new crown swab samples.
TABLE 9 formulation of nucleic acid extraction kit of this example
EXAMPLE 3 kits of the invention
The system of the kit of the invention is shown in Table 10 for African swine fever oral-nasal swab samples.
TABLE 10 formulation of nucleic acid extraction kit of this example
Example 4 comparison of the extraction of the kit according to the invention with the prior art kit
Based on the optimal concentration ratio range of the mass of CTAB and N-lauroyl sarcosine sodium, the optimal mass concentration is optimized, and compared with a control reagent containing a proteinase K system.
The invention aims at the comparison of the extraction efficiency and the extraction time of two samples, namely the system of the invention and a control reagent system.
In this example, the performance of the nucleic acid extraction or purification kit of the present invention was verified, and samples containing novel coronaviruses and african swine fever viruses were extracted using the kit of the present invention, and compared with the extraction effect of a control kit (the nucleic acid extraction kit (Ex-DNA/RNA virus) of siennan technologies, inc.) and the performance of the kit of the present invention was verified by the control reagent extraction procedure shown in table 11. The composition of the control kit (Seamanlon technology Co., ltd. Nucleic acid extraction kit (Ex-DNA/RNA virus)) is shown in Table 12.
Table 11, control reagent GeneRotex96 Autoextractor extraction program
TABLE 12 nucleic acid extraction kit (Ex-DNA/RNA Virus) Components of the Seaman technology Co., ltd
The verification method comprises the following steps: the average Ct value and CV value after 4 times of extraction were calculated by using the kit system of the present invention of example 2 and example 3, and the control kit system, respectively, using the same new crown swab sample and African swine fever sample for 4 times of extraction. The results show that for the new crown swab samples, the extraction efficiency is better and the extraction time is shorter compared with the control kit system containing proteinase K, and the results are shown in Table 13.
For African swine fever oronasal swab samples, the kit system of the invention has better extraction efficiency and shorter extraction time compared with the control kit system containing proteinase K, and the results are shown in Table 14.
Table 13 comparison of sample data from extraction of new crown swabs from the inventive and control kits
Table 14 comparison of african swine fever sample data extracted from the kit of the present invention with the control kit
In summary, example 4 verifies that the kit system of the invention can realize nucleic acid extraction for different extraction objects and sample types, the extraction efficiency is basically advanced compared with a control kit, and compared with the control kit, the kit of the invention has short extraction time, does not need proteinase K, and has simpler operation.
Comparative example
The combination of surfactants was varied on the basis of example 3, in comparison with the combination of the substances N-lauroyl sarcosine sodium and CTAB according to the invention, wherein comparative example 1 is a combination of CTAB and lithium dodecyl sulfate, comparative example 2 is a combination of Triton X-100 and N-lauroyl sarcosine sodium, and comparative example 3 is a combination of Triton X-10 and lithium dodecyl sulfate. The extraction effect of the present invention and comparative examples 1 to 3 was verified.
The verification method comprises the following steps: the same african swine fever sample was extracted 4 times using the kit system of example 3 of the present invention and the kit systems of comparative examples 1 to 3, respectively, and the average value of Ct values and CV value after 4 times of extraction were calculated. The results show that compared with the comparative kit system compounded by different surfactants, the kit system has better extraction efficiency for African swine fever samples, and the results are shown in Table 15.
TABLE 15 comparison of African swine fever sample data extracted from the inventive example 3 kit with the comparative examples 1-3 kit
The above is an embodiment exemplified in this example, but this example is not limited to the above-described alternative embodiments, and a person skilled in the art may obtain various other embodiments by any combination of the above-described embodiments, and any person may obtain various other embodiments in the light of this example. The above detailed description should not be construed as limiting the scope of the present embodiments, which is defined in the claims and the description may be used to interpret the claims.
Claims (10)
1. A kit for nucleic acid extraction or purification without the need for proteinase K, characterized in that: comprises a lysate, wherein the lysate comprises 1.0 mol/L-5.0 mol/L guanidine isothiocyanate, 10 mmol/L-50 mmol/L sodium acetate, 10 mmol/L-30 mmol/L EDTA,0.5 mol/L-5.0 mol/L NaCl, urea with the mass percentage of 0.1-3 percent, isopropanol with the volume ratio of 10-70 percent, CTAB and N-lauroyl sodium sarcosinate, wherein the mass concentration ratio of the CTAB to the N-lauroyl sodium sarcosinate is 10-50.
2. A proteinase K-free nucleic acid extraction or purification kit according to claim 1, wherein: the concentration of CTAB is 10 mmol/L-30 mmol/L, and the concentration of N-lauroyl sarcosine sodium is 0.5 mmol/L-2 mmol/L.
3. A proteinase K-free nucleic acid extraction or purification kit according to claim 2, characterized in that: for the new crown swab samples, the concentration of CTAB and N-lauroyl sarcosinate was 20mM and 1.5mM, respectively; for oronasal swab samples, the concentration of CTAB and N-lauroyl sarcosinate was 15mM and 1.0mM, respectively.
4. A proteinase K-free nucleic acid extraction or purification kit according to claim 1, wherein: for a new crown swab sample, the lysate comprises 3mol/L guanidine isothiocyanate, 30mmol/L sodium acetate, 10mmol/L EDTA, 3mol/L NaCl, urea with the mass percent of 2% and isopropanol with the volume ratio of 20%; for African swine fever samples, the lysate comprises 3.5mol/L guanidine isothiocyanate, 30mmol/L sodium acetate, 10mmol/L EDTA, 3mol/L NaCl, 2% urea by mass and 20% isopropanol by volume.
5. A proteinase K-free nucleic acid extraction or purification kit according to claim 1, wherein: the kit also comprises a washing liquid 1, wherein the washing liquid 1 comprises 10-50 mmol/L of sodium acetate, 5-30 mmol/L of EDTA, 0.5-3.0 mol/L of NaCl and 30-70% of isopropanol or absolute ethyl alcohol by volume ratio.
6. The proteinase K-free nucleic acid extraction or purification kit of claim 5, wherein: the washing liquid 1 comprises 15mmol/L sodium acetate, 20mmol/L EDTA,0.8mol/L NaCl and 50% isopropyl alcohol by volume.
7. A proteinase K-free nucleic acid extraction or purification kit according to claim 1, wherein: the kit also comprises a washing liquid 2, wherein the washing liquid 2 comprises 0.5-3 mmol/L of sodium citrate, 1-10 mmol/L of EDTA and 40-90% of isopropanol or absolute ethyl alcohol by volume ratio.
8. The proteinase K-free nucleic acid extraction or purification kit of claim 7, wherein: the washing liquid 2 comprises 2.0mmol/L sodium citrate, 1.0mmol/L EDTA and 60% absolute ethyl alcohol by volume.
9. A proteinase K-free nucleic acid extraction or purification kit according to claim 1, wherein: the kit also comprises an eluent, wherein the eluent comprises 0.1 mmol/L-10 mmol/L Tris.
10. A proteinase K-free nucleic acid extraction or purification kit according to claim 1, wherein: the kit also comprises a magnetic bead mixed solution, wherein the magnetic bead mixed solution comprises magnetic beads and a magnetic bead diluent, the functional groups carried by the magnetic beads comprise but are not limited to amino groups, hydroxyl groups, carboxyl groups or silicon hydroxyl groups, and the concentration of the magnetic beads in the magnetic bead mixed solution is 15mg/ml.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118109459A (en) * | 2024-04-30 | 2024-05-31 | 苏州英泽生物医药科技有限公司 | Kit for extracting RNA (ribonucleic acid) by magnetic bead method without proteinase k treatment and extraction method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118109459A (en) * | 2024-04-30 | 2024-05-31 | 苏州英泽生物医药科技有限公司 | Kit for extracting RNA (ribonucleic acid) by magnetic bead method without proteinase k treatment and extraction method |
| CN118109459B (en) * | 2024-04-30 | 2024-06-25 | 苏州英泽生物医药科技有限公司 | Kit for extracting RNA (ribonucleic acid) by magnetic bead method without proteinase k treatment and extraction method |
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