CN115960885B - Method and composition for extracting nucleic acid from heparin sodium sample - Google Patents
Method and composition for extracting nucleic acid from heparin sodium sample Download PDFInfo
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- 239000000203 mixture Substances 0.000 title claims abstract description 46
- 229920000669 heparin Polymers 0.000 title claims abstract description 35
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 32
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 32
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 25
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 title claims abstract description 19
- 229960001008 heparin sodium Drugs 0.000 title claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000006166 lysate Substances 0.000 claims abstract description 50
- 239000007788 liquid Substances 0.000 claims description 52
- 238000005406 washing Methods 0.000 claims description 41
- 239000000243 solution Substances 0.000 claims description 22
- 239000007983 Tris buffer Substances 0.000 claims description 20
- 238000001179 sorption measurement Methods 0.000 claims description 19
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 18
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 18
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 18
- 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 15
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 15
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 229920004890 Triton X-100 Polymers 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 8
- RPACBEVZENYWOL-XFULWGLBSA-M sodium;(2r)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate Chemical compound [Na+].C=1C=C(Cl)C=CC=1OCCCCCC[C@]1(C(=O)[O-])CO1 RPACBEVZENYWOL-XFULWGLBSA-M 0.000 claims description 7
- 239000013504 Triton X-100 Substances 0.000 claims description 6
- 108020000999 Viral RNA Proteins 0.000 claims description 6
- 230000009089 cytolysis Effects 0.000 claims description 6
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 15
- 238000000605 extraction Methods 0.000 abstract description 15
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 15
- 230000003612 virological effect Effects 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 40
- 238000011529 RT qPCR Methods 0.000 description 28
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 25
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 16
- 229960002897 heparin Drugs 0.000 description 16
- 238000001514 detection method Methods 0.000 description 15
- 230000000694 effects Effects 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 10
- 229960005486 vaccine Drugs 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 229910021645 metal ion Inorganic materials 0.000 description 8
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 7
- 239000002738 chelating agent Substances 0.000 description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000003398 denaturant Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000001509 sodium citrate Substances 0.000 description 6
- 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 description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 6
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 5
- 229960003237 betaine Drugs 0.000 description 5
- 239000012928 buffer substance Substances 0.000 description 5
- 239000004094 surface-active agent Substances 0.000 description 5
- 108010022901 Heparin Lyase Proteins 0.000 description 4
- -1 mops) Substances 0.000 description 4
- 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 3
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 3
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 3
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 229960003964 deoxycholic acid Drugs 0.000 description 3
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 3
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 3
- 229920000053 polysorbate 80 Polymers 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 description 3
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 3
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 229920001661 Chitosan Polymers 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 108700005077 Viral Genes Proteins 0.000 description 2
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- 238000009472 formulation Methods 0.000 description 2
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- 239000002245 particle Substances 0.000 description 2
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- 239000011734 sodium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 208000001435 Thromboembolism Diseases 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
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- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
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Abstract
The present disclosure provides a nucleic acid extraction method and a composition, belongs to the field of biotechnology, and in particular relates to a method for extracting viral nucleic acid from heparin sodium samples and a composition thereof. The methods of the present disclosure effectively improve nucleic acid amplification efficiency by reducing ethanol content in the lysate and/or wash liquor.
Description
Technical Field
The disclosure belongs to the field of nucleic acid extraction, and in particular relates to a method for extracting nucleic acid from a heparin sodium sample and a composition thereof.
Background
Heparin is used as an anticoagulant and is a polymer formed by alternately connecting two polysaccharides, and has an anticoagulant effect in vivo and in vitro. Can be used for treating thromboembolic diseases, myocardial infarction, cardiovascular operation, heart catheter examination, extracorporeal circulation, hemodialysis, etc. Meanwhile, heparin is used as a strong inhibitor of PCR, has similar characteristics to nucleic acid, and can be combined with a polymerase active site to inhibit PCR. Therefore, biological samples such as heparin-containing blood, plasma and the like need to be subjected to different pretreatment methods to remove the influence caused by heparin before PCR, so that false negative is prevented, or patients are required to terminate heparin treatment in advance, and the heparin content in the body is reduced. The first method for removing heparin is digestion by using heparinase, the method is long in time consumption, commercial heparinase is high in price, and Langer et al report that heparinase system is used for in-vitro treatment100% heparin removal was achieved by enzymatic degradation using a heparinase immobilized hemofilter (Langer R, linhardt RJ, cooney CL, klein M, tapper D, hoffberg SM, larsenA.1982.An enzymatic system for removing heparin in extracorporeal therapy.science.217:261-263). The second is the use of some mediator materials to adsorb heparin in biological fluids, as reported by Kaminski et al, using a genipin cross-linked chitosan microsphere sensitive to pH to remove heparin from water with high efficiency, and future applications in biomedical fields (Kaminski K, zazakowny K, szczubiaka K, nowakowska M. PH-Sensitive genipin-cross-linked chitosan microspheres for heparin removal. Biomacromolecules.2008; 9:3127-3132), as well asBaydemir et al reported the removal of heparin from plasma using molecularly imprinted cryogels. The third is a method of nucleic acid purification, which removes heparin impurities during the purification process. The method for extracting genome from heparin product as shown in CN103952398A takes only 40min, and compared with the method, the method has the advantages of greatly reduced time cost. In addition, the first two methods are high in cost and time consuming, unique materials cannot be widely used, and only heparin in a sample is removed by the two methods, and an additional nucleic acid extraction kit is needed if nucleic acid in the sample is needed.
Disclosure of Invention
The purpose of the present disclosure is to propose a method that can extract viral nucleic acid from heparin sodium plasma sample rapidly, by specific lysis, binding, washing, rinsing system, remove various impurities contained in the sample, especially heparin sodium. The extracted product can be directly applied to various downstream applications such as PCR and the like, can be compatible with various liquid samples including heparin sodium samples, and provides convenience for clinical application.
In a first aspect, the present disclosure provides a liquid composition comprising a buffer substance, a denaturant, a surfactant, and absolute ethanol, wherein the absolute ethanol has a volume fraction of 20% -35%. The liquid composition is used for cleaving viral particles in a plasma sample containing heparin sodium, releasing viral DNA and/or RNA nucleic acids.
In some embodiments, the volume fraction of the absolute ethanol is 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%.
In some embodiments, the buffer substance is selected from at least one of tris, 3- (N-morpholino) propanesulfonic acid (i.e., mops), sodium acetate, sodium citrate, sodium carbonate.
In some embodiments, the denaturant is selected from at least one of guanidine isothiocyanate, guanidine hydrochloride, guanidine thiocyanate, perchlorate, naI, KI, urea.
In some embodiments, the surfactant is selected from at least one, at least two, or at least three of Triton X-100, tween-20, tween-80, NP-40, SDS, CHAPS, sodium deoxycholate, sodium cholate, betaine, PEG 8000.
In some embodiments, the liquid composition comprises tris, guanidinium isothiocyanate, tween-20, triton X-100 and absolute ethanol, wherein the volume fraction of absolute ethanol is 20% -35%. In some embodiments, the tris concentration is 20mM, the guanidine isothiocyanate concentration is 2M, the tween-20 volume fraction is 1%, the Triton X100 volume fraction is 0.1%, and the absolute ethanol volume fraction is 24% -32%. In some embodiments, the volume fraction of the absolute ethanol is 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%.
In some embodiments, the liquid composition further comprises a metal ion chelating agent. In some embodiments, the metal ion chelating agent is selected from at least one of edta.2na, edta.4na, EDTA, sodium citrate, EGTA, HEDTA. In some embodiments, the metal ion chelating agent is EDTA-2 Na at a concentration of 5mM.
In some embodiments, the liquid composition further comprises betaine at a concentration of 0.1mM.
In some embodiments, the liquid composition further comprises PEG8000, the volume fraction of PEG8000 being 0.005%.
In some embodiments, the pH of the liquid composition is from 7.0 to 7.5. In some embodiments, the pH of the liquid composition is 7.3.
In a second aspect, the present disclosure provides a liquid composition comprising a buffer substance, a metal ion chelating agent, a denaturant, a surfactant, and absolute ethanol, wherein the volume fraction of absolute ethanol is 15% -45%. The liquid composition is used for removing impurities such as residual proteins.
In some embodiments, the absolute ethanol volume fraction is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%.
In some embodiments, the buffer substance is selected from at least one of tris, 3- (N-morpholino) propanesulfonic acid (i.e., mops), sodium acetate, sodium citrate, sodium carbonate.
In some embodiments, the metal ion chelating agent is selected from at least one of edta.2na, edta.4na, EDTA, sodium citrate, EGTA, HEDTA. In some embodiments, the metal ion chelating agent is EDTA-2 Na at a concentration of 10mM.
In some embodiments, the denaturant is selected from at least one of guanidine isothiocyanate, guanidine hydrochloride, guanidine thiocyanate, perchlorate, naI, KI, urea.
In some embodiments, the surfactant is selected from at least one, at least two, or at least three of Triton X-100, tween-20, tween-80, NP-40, SDS, CHAPS, sodium deoxycholate, sodium cholate.
In some embodiments, the liquid composition comprises tris, EDTA-2 Na salt, guanidine hydrochloride, tween-20, and absolute ethanol, wherein the volume fraction of absolute ethanol is 15% -45%. In some embodiments, the tris concentration is 20mM, EDTA-2 Na salt concentration is 10mM, guanidine hydrochloride concentration is 2M, tween-20 volume fraction is 0.002%, and the volume fraction of absolute ethanol is 15% -45%. In some embodiments, the absolute ethanol volume fraction is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%.
In some embodiments, the liquid composition has a pH of 7.3 to 7.7. In some embodiments, the pH of the liquid composition is 7.5.
In a third aspect, the present disclosure provides a kit comprising the liquid composition of the first aspect and/or the liquid composition of the second aspect.
In some embodiments, the kit further comprises a liquid composition for rinsing the adsorption column, the liquid composition comprising tris, EDTA-2 Na salt, guanidine hydrochloride, sodium chloride, and ethanol. In some embodiments, the tris concentration is 20mM, EDTA-2 Na salt concentration is 0.5mM, guanidine hydrochloride concentration is 100mM, sodium chloride concentration is 50mM, ethanol volume fraction is 80%. In some embodiments, the liquid composition used to rinse the adsorption column has a pH of 8.0.
In some embodiments, the kit further comprises a liquid composition for eluting nucleic acids, the liquid composition comprising tris and nulease-free Water. In some embodiments, the tris concentration is 10mM and the liquid composition pH is 6.0.
In a fourth aspect, the present disclosure provides the use of a kit according to the third aspect for extracting viral RNA from a heparin sodium containing sample. In some embodiments, there is provided the use of a kit according to the third aspect for co-extracting DNA and RNA nucleic acids on a heparin sodium containing sample.
In a fifth aspect, the present disclosure provides a method of sample nucleic acid extraction, the method comprising sample lysis, nucleic acid binding adsorption column, washing adsorption column, rinsing adsorption column, eluting nucleic acid, wherein the sample is lysed using the liquid composition of the first aspect described above and/or the adsorption column is washed using the liquid composition of the second aspect described above.
In some embodiments, the sample is a plasma sample containing heparin sodium.
In some embodiments, the sample nucleic acid extraction method is used to extract viral RNA in a sample. In some embodiments, the sample nucleic acid extraction method is used to co-extract DNA and RNA in a sample.
The method disclosed by the invention can effectively enrich virus nucleic acid, has a better heparin sodium removal effect, and reduces the influence of heparin sodium on the amplification efficiency of PCR or QPCR. The extracted product can be directly applied to downstream reverse transcription, PCR, fluorescent quantitative PCR, RT-qPCR, second generation sequencing, biochip analysis and the like.
Name interpretation
"buffer substance" herein refers to a substance capable of maintaining a solution pH environment, including tris, 3- (N-morpholino) propanesulfonic acid (i.e., mops), sodium acetate, sodium citrate, sodium carbonate, and the like.
As used herein, "denaturant" refers to a denaturant that has the effect of cleaving viral particles, denaturing proteins, inhibiting RNase, providing a high salt environment, and is capable of promoting binding of viral DNA and/or RNA to an adsorption membrane, including guanidine isothiocyanate, guanidine hydrochloride, guanidine thiocyanate, perchlorate, naI, KI, urea, and the like.
As used herein, "surfactant" refers to a compound that has the effect of solubilizing, denaturing, or otherwise denaturing proteins in a sample, including Triton X-100, tween-20, tween-80, NP-40, SDS, CHAPS, sodium deoxycholate, sodium cholate, betaine, PEG8000, and the like.
Herein, "metal ion chelating agent" means a substance having a metal ion chelating effect, and includes EDTA.2Na, EDTA.4Na, EDTA, sodium citrate, EGTA, HEDTA, and the like.
Drawings
Fig. 1: extracting PEDV nucleic acid qPCR results from human plasma by different lysates;
fig. 2: extracting PRV nucleic acid qPCR results from human plasma by different lysates;
fig. 3: qPCR results of extracting PEDV nucleic acid in human plasma from three lysates in example 2;
fig. 4: the three washes of example 3 extract PEDV nucleic acid qPCR results from human plasma;
fig. 5: the two washes of example 4 extract PEDV nucleic acid qPCR results from human plasma;
fig. 6: qPCR results of PEDV nucleic acid in human plasma were extracted from two different combinations in example 5.
Detailed description of the preferred embodiments (examples)
The following embodiments are further described with reference to the drawings, but the following examples are merely simple examples of the disclosure and do not represent or limit the scope of the claims of the disclosure.
In the following examples, reagents and consumables were purchased from manufacturers of reagents conventional in the art unless specifically stated otherwise; unless otherwise indicated, all methods and techniques used are those conventional in the art.
Example 1: extracting virus samples containing heparin sodium from different lysates and detecting
1. Analog sample preparation
Taking 10 parts of kefujing vaccine (containing porcine epidemic diarrhea virus PEDV vaccine) (keqiong ) and 10 parts of keqining vaccine (containing pseudorabies virus PRV vaccine) (keqiong ), respectively adding 10ml of keqiong special vaccine diluent and keqiong special vaccine diluent, fully dissolving and uniformly mixing, and using PBS to respectively and gradiently dilute the two dissolved vaccines by 100 times. 2 μl of diluted Corning vaccine and 2 μl of diluted Corning vaccine are added into 296 μl of human heparin sodium plasma sample, and mixed thoroughly to obtain a simulated nucleic acid extraction sample. Sample preparation was performed in the above scale up with the actual required sample size for each experiment.
2. Simulated sample lysis
20 μl PK, 300 μl of the simulated sample and 500 μl of lysate of different components were sequentially added to three groups of 1.5ml centrifuge tubes, respectively, and incubated at room temperature for 5min after thoroughly shaking and mixing.
Different lysates were prepared according to table 1:
table 1: different components and contents of the lysate
| Component (A) | Lysate 1 | Lysate 2 | Lysate 3 |
| Trimethylolaminomethane | 20mM | 20mM | 20mM |
| EDTA·2Na·2H 2 O | 5mM | 5mM | / |
| Guanidine isothiocyanate | 2M | 2M | 2M |
| Betaine (betaine) | 0.1mM | / | / |
| Tween-20 | 1％ | 1％ | 1％ |
| TritonX-100 | 0.1％ | 0.1％ | 0.1％ |
| PEG8000 | 0.005％ | 0.005％ | / |
| Absolute ethyl alcohol | 32％ | 32％ | 32％ |
| pH | 7.3 | 7.3 | 7.3 |
3. Nucleic acid binding adsorption column
And (3) transferring all the three groups of incubated cracking samples into three groups of adsorption columns respectively, centrifuging at 12000rpm for 1min, and discarding the waste liquid.
4. Washing adsorption column
To each of the three adsorption columns, 700. Mu.l of the washing solution was added, and the mixture was centrifuged at 12000rpm for 30sec to discard the waste liquid. The washing liquid comprises the following components in percentage by weight: 20mM tris (hydroxymethyl) aminomethane, 10mM EDTA.2Na.2H 2 O, 2M guanidine hydrochloride, 0.002% Tween-20 and 40% absolute ethanol, and the pH of the washing solution is 7.5 (the washing solution with the ratio is named as washing solution 1).
5. Rinsing adsorption column
To the washed adsorption column, 700. Mu.l of the rinse solution was added, and the mixture was centrifuged at 12000rpm for 30sec, whereby the waste liquid was discarded. The rinse solution used therein comprises the following components in percentage by weight: 20mM tris (hydroxymethyl) aminomethane, 0.5mM EDTA.2Na.2H 2 O, 100mM guanidine hydrochloride, 50mM sodium chloride, 80% absolute ethanol, and pH8.0 (the ratio of the rinse solution is designated as rinse solution 1).
6. Eluting the cleavage product
Firstly, putting the adsorption column in the step of centrifugation at 12000rpm for 2min, transferring the adsorption column into a new 1.5mL centrifuge tube, adding 50 μl of eluent, standing for 1min, and centrifuging at 12000rpm for 1min to obtain three solutions which are virus DNA/RNA solutions extracted from the simulated sample. Wherein the eluent composition and the content of the eluent are 10mM of trimethylol aminomethane, nucleic-free Water and pH6.0.
7. Detection of the extracted product
Using HiScript II U + The extract was subjected to qPCR detection using One Step qRT-PCR Probe Kit (Nuo-vozan organism, cat# Q222-CN).
The extracted products were subjected to PEDV and PRV virus gene detection according to the reaction system of table 2 and the reaction procedure of table 3.
Table 2: qPCR reaction system
| RNase-freeddH 2 O | 9.4μl |
| 2×OneStepU + Mix | 15μl |
| OneStepU + EnzymeMix | 1.5μl |
| 50×ROXReferenceDye2 | 0.6μl |
| PEDV-F or PRV-F (10. Mu.M) | 0.6μl |
| PEDV-R or PRV-R (10. Mu.M) | 0.6μl |
| PEDV-Probe or PRV-Probe (10. Mu.M) | 0.3μl |
| Extracting the product | 2μl |
| Total volume of | 30μl |
Table 3: qPCR reaction procedure
The primer probe sequences used for the detection of PEDV and PRV viral genes are shown in Table 4.
Table 4: primer probe sequence for detecting PEDV and PRV virus genes
| Sequence numbering | Sequence name | Sequence(s) |
| SEQ ID NO:1 | PEDV-F | CGTGAGCCTGGCTTAGTCTTG |
| SEQ ID NO:2 | PEDV-R | CATACGTCGCGATGAAACAAA |
| SEQ ID NO:3 | PEDV-Probe | CGCATGAACTTCAAAATCATACTGCGACG |
| SEQ ID NO:4 | PRV-F | GCGTGGACCAGCACCG |
| SEQ ID NO:5 | PRV-R | TCCACGCCCCGCTTG |
| SEQ ID NO:6 | PRV-Probe | CAAGTTCGGCGCGTGCTGGA |
8. Analysis of results
qPCR results are shown in fig. 1 (PEDV virus detection result) and fig. 2 (PRV virus detection result), wherein L1 represents qPCR results of obtaining a product by lysing a dummy sample with lysate 1, L2 represents qPCR results of obtaining a product by lysing a dummy sample with lysate 2, and L3 represents qPCR results of obtaining a product by lysing a dummy sample with lysate 3. Figure 1 shows that three lysates of different formulations are used for extracting PEDV virus with comparable extraction effects, and figure 2 shows that three lysates of different formulations are used for extracting PRV virus with comparable extraction effects.
Example 2: reducing the ethanol content in the lysate can effectively improve the amplification efficiency of the extracted product
Lysate 3, lysate 4 and lysate 5 were prepared according to table 5, and the simulated samples were subjected to viral nucleic acid lysis extraction using lysate 3, lysate 4 and lysate 5, respectively, to obtain three extracted products, and qPCR viral gene detection was performed on the three products, and the effects of the three lysates on the extracted products were compared.
Table 5: lysate 3, lysate 4 and lysate 5 components and content
| Component (A) | Lysate 3 | Lysate 4 | Lysate 5 |
| Trimethylolaminomethane | 20mM | 20mM | 20mM |
| Guanidine isothiocyanate | 2M | 2M | 2M |
| Tween-20 | 1％ | 1％ | 1％ |
| TritonX-100 | 0.1％ | 0.1％ | 0.1％ |
| Absolute ethyl alcohol | 32％ | 24％ | 40％ |
| pH | 7.3 | 7.3 | 7.3 |
Wherein the simulated sample preparation, the rinsing liquid, the eluent and the qPCR reaction in the present example are the same as those in example 1, the washing liquid used in the present example comprises 20mM tris, 10mM EDTA.2Na.2H 2 O, 2M guanidine hydrochloride, 0.002% Tween-20 and 57% absolute ethanol, and the pH of the washing solution is 7.5 (the washing solution with the ratio is named as washing solution 2).
Analysis of results
Example 2 the results are shown in FIG. 3, wherein L3, L4, L5 represent the results of the PEDV gene detection qPCR on the extracted products obtained using lysate 3, lysate 4, lysate 5, respectively. From fig. 3, it can be seen that the effects of extracting PEDV virus by using the lysate 3 and the lysate 4 are equivalent, and the effect of extracting PEDV virus by using the lysate 5 is the worst, which indicates that the amplification efficiency of PEDV extraction products can be effectively improved by reducing the ethanol content in the lysate.
Example 3: effect of three different washes on amplification efficiency of extracted product
Washing liquid 2, washing liquid 3 and washing liquid 4 were prepared according to table 6, and the simulation samples were extracted using washing liquid 2, washing liquid 3 and washing liquid 4, respectively, to obtain three extracted products, and qPCR virus gene detection was performed on the three products, and the influence of the three washing liquids on the extracted products was compared.
Table 6: washing liquid 2, washing liquid 3 and washing liquid 4 components and content
| Component (A) | Washing liquid 2 | Washing liquid 3 | Washing liquid 4 |
| Trimethylolaminomethane | 20mM | 20mM | 20mM |
| EDTA·2Na·2H 2 O | 10mM | 10mM | 10mM |
| Guanidine hydrochloride | 2M | 2M | 2M |
| Tween-20 | 0.002％ | 0.002％ | 0.002％ |
| Absolute ethyl alcohol | 57％ | 28％ | 17％ |
| pH | 7.5 | 7.5 | 7.5 |
The simulated sample preparation, the rinse, the eluent, and the qPCR reaction in this example were all the same as in example 1, and the lysate used in this example was lysate 5 in example 2.
Analysis of results
Example 3 the results are shown in FIG. 4, wherein W2, W3, W4 represent the results of the PEDV gene detection qPCR on the extracted products obtained using washing liquid 2, washing liquid 3, washing liquid 4, respectively. From fig. 4, it can be seen that the amplification efficiency of extracting PEDV virus by using the washing liquid 3 and the washing liquid 4 is equivalent, while the amplification efficiency of extracting the product by using the washing liquid 2 is slightly lower, which indicates that the amplification efficiency of extracting the product by PEDV can be effectively improved by reducing the ethanol content in the washing liquid.
Example 4: effect of two different washes on amplification efficiency of extracted product
Washing solutions 1 and 2 were prepared according to examples 1 and 3, respectively, and the simulation samples were extracted using washing solutions 1 and 2, respectively, to obtain two extracted products, and qPCR virus gene detection was performed on the two products, and the effect of the two washing solutions on amplification efficiency of the extracted products was compared. The simulated sample preparation, the rinse, the eluent, and the qPCR reaction in this example were all the same as in example 1, and the lysate used in this example was lysate 5 in example 2.
Analysis of results
Example 4 the results are shown in FIG. 5, wherein W1 and W2 represent the results of qPCR for PEDV gene detection on the extracted products obtained using washing solutions 1 and 2, respectively. From fig. 5, it can be seen that the amplification efficiency of PEDV virus extraction using the washing solution 1 is significantly better than that using the washing solution 2, which indicates that the reduction of ethanol content in the washing solution can effectively improve PEDV product quality.
Example 5: simultaneously, the content of ethanol in the lysate and the washing liquid is reduced, so that the amplification efficiency of the extracted product can be effectively improved
Nucleic acid extraction was performed on the simulated biological sample using lysate 5 and wash 2 (designated as combination 1), nucleic acid extraction was performed on the simulated biological sample using lysate 4 and wash 1 (designated as combination 2), qPCR virus gene detection was performed on the two nucleic acid extracts, and the effect of the two combinations on the amplification efficiency of the extracted products was compared. The simulated sample preparation, the rinse, the eluent, and the qPCR reaction in this example were the same as in example 1.
Analysis of results
Example 5 the results are shown in fig. 6, wherein Before represents the result of PEDV gene detection qPCR on the extract product obtained using combination 1, and After represents the result of PEDV gene detection qPCR on the extract product obtained using combination 2. From fig. 6, it can be seen that the amplification efficiency of PEDV extraction using combination 2 is significantly better than that of combination 1, demonstrating that reducing the ethanol content in the lysate and wash liquor can effectively increase the amplification efficiency of PEDV products.
Claims (9)
1. Use of a kit for extracting viral RNA or co-extracting DNA and RNA nucleic acids on a heparin sodium containing sample, the kit comprising a lysate composition comprising tris, guanidine isothiocyanate, tween-20, triton X-100 and 24% -32% by volume absolute ethanol and/or a wash composition comprising tris, EDTA-2 Na salt, guanidine hydrochloride, tween-20 and 17% -40% by volume absolute ethanol.
2. The use of a kit according to claim 1, wherein the volume fraction of absolute ethanol in the lysate composition is 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31% or 32%, and the volume fraction of absolute ethanol in the wash composition is 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%.
3. The use of the kit of claim 1, wherein the composition of the lysate comprises 20mM tris, 2M guanidine isothiocyanate, 1% tween-20 and 0.1% Triton X100, and the composition of the wash comprises 20mM tris, 10mM edta-2 Na salt, 2M guanidine hydrochloride and 0.002% tween-20.
4. Use of a kit according to any one of claims 1-3, wherein the lysate composition has a pH of 7.3 and the wash liquor composition has a pH of 7.5.
5. The use of a kit according to claim 1, further comprising a rinse solution composition comprising tris, edta.2na salt, guanidine hydrochloride, sodium chloride and ethanol.
6. The use of a kit according to claim 1, further comprising an eluent composition comprising tris and nulease-free Water.
7. A method of extracting viral RNA or co-extracting DNA and RNA nucleic acids on a heparin sodium sample, the method comprising sample lysis, nucleic acid binding adsorption column, washing adsorption column, rinsing adsorption column, eluting nucleic acids, wherein the sample lysis step is accomplished by a lysate composition comprising tris, guanidinium isothiocyanate, tween-20, triton X-100 and 24% -32% absolute ethanol by volume fraction.
8. The method of claim 7, wherein the wash adsorption column is completed with a wash liquid composition comprising tris, EDTA-2 Na salt, guanidine hydrochloride, tween-20 and 17-40% absolute ethanol by volume.
9. The method of any one of claims 7 or 8, wherein the heparin sodium sample is a plasma sample containing heparin sodium.
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