CN115960885A - 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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- CN115960885A CN115960885A CN202211225861.0A CN202211225861A CN115960885A CN 115960885 A CN115960885 A CN 115960885A CN 202211225861 A CN202211225861 A CN 202211225861A CN 115960885 A CN115960885 A CN 115960885A
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- 239000000203 mixture Substances 0.000 title claims abstract description 62
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 36
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 36
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 36
- 229920000669 heparin Polymers 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 21
- 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 17
- 229960001008 heparin sodium Drugs 0.000 title claims abstract description 15
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- 238000005406 washing Methods 0.000 claims abstract description 45
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 18
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- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound 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 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims 2
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- 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 15
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Images
Abstract
The disclosure provides a nucleic acid extraction method and a composition, belongs to the technical field of biology, and particularly relates to a method for extracting viral nucleic acid from a heparin sodium sample and a composition thereof. The method disclosed by the invention effectively improves the nucleic acid amplification efficiency by reducing the ethanol content in the lysis solution and/or the washing solution.
Description
Technical Field
The disclosure belongs to the field of nucleic acid extraction, and particularly relates to a method for extracting nucleic acid from a heparin sodium sample and a composition thereof.
Background
Heparin, as an anticoagulant, is a polymer formed by alternately connecting two polysaccharides and has anticoagulant effects both inside and outside the body. Can be clinically used for treating various diseases such as thromboembolic diseases, myocardial infarction, cardiovascular operations, cardiac catheter examination, extracorporeal circulation, hemodialysis and the like. Meanwhile, heparin, as a recognized strong PCR inhibitor, has the similar characteristics with nucleic acid and can be combined with the active site of polymerase to inhibit PCR. Therefore, the biological samples such as blood, plasma and the like containing heparin are subjected to PCRDifferent pretreatment methods are needed to remove the influence caused by heparin so as to prevent false negative, or the patient is required to stop heparin treatment in advance so that the content of heparin in the body of the patient is reduced. The first of the current methods for heparin removal is digestion with heparinase, which is time consuming and expensive commercially available, langer et al report the use of the heparinase system for extracorporeal treatment, the use of heparinase to immobilize blood filters, and the 100% removal of heparin by enzymatic degradation (Langer R, linhardt RJ, cooney CL, klein M, tapper D, hoffberg SM, larsen A.1982.An enzymatic system for removing heparin in exogenous hearing therapy.217: 261-263). The second is the use of some media materials for adsorbing heparin in biological fluids, such as Kaminski et al reported that heparin can be efficiently removed from water using a pH Sensitive genipin cross-linked chitosan microsphere, which is likely to be applied in the biomedical field in the future (Kaminski K, zazakowny K, szczubialka K, nowakowska M. PH-Sensitive gene-cross-linked chitosan microspheres for biomedical devices 2008;9
Baydemir et al reported the removal of heparin from plasma using a molecularly imprinted cryogel. The third is a method for nucleic acid purification, which can remove heparin impurities during the purification process. The method for extracting genome from heparin products, as shown in CN103952398A, takes only 40min as a whole, and compared with the method, the time cost is greatly reduced. In addition, the first two methods are high in cost and long in time consumption, unique materials cannot be widely applied, heparin in the sample is only removed, and if nucleic acid in the sample needs to be extracted, an additional nucleic acid extraction kit is needed.
Disclosure of Invention
The purpose of the present disclosure is to provide a method for rapidly extracting viral nucleic acid from a heparin sodium plasma sample, which removes various impurities contained in the sample, particularly heparin sodium, through a specific lysis, binding, washing and rinsing system. The extracted product can be directly applied to various downstream applications such as PCR and the like, and simultaneously can be compatible with various liquid samples containing heparin sodium samples, thereby providing convenience for clinical application.
In a first aspect, the present disclosure provides a liquid composition comprising a buffer substance, a denaturant, a surfactant, and anhydrous ethanol, wherein the volume fraction of the anhydrous ethanol is 20% to 35%. The liquid composition is used for lysing virus particles in a plasma sample containing heparin sodium, and releasing virus DNA and/or RNA nucleic acid.
In some embodiments, the volume fraction of the anhydrous 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-morpholine) propanesulfonic acid (i.e., mops), sodium acetate, sodium citrate, sodium carbonate.
In some embodiments, the denaturant is selected from at least one of guanidinium isothiocyanate, guanidinium hydrochloride, guanidinium 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 between 20% and 35%. In some embodiments, the tris concentration is 20mM, the guanidinium 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% to 32%. In some embodiments, the volume fraction of the anhydrous 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 the PEG8000 being 0.005%.
In some embodiments, the liquid composition has a pH of 7.0 to 7.5. In some embodiments, the liquid composition has a pH of 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 anhydrous ethanol, wherein the volume fraction of the anhydrous ethanol is 15% to 45%. The liquid composition is used for removing impurities such as residual protein.
In some embodiments, the anhydrous 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-morpholine) 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 guanidinium isothiocyanate, guanidinium hydrochloride, guanidinium 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% to 45%. In some embodiments, the tris concentration is 20mM, the edta.2na salt concentration is 10mM, the guanidine hydrochloride concentration is 2M, the tween-20 volume fraction is 0.002%, and the absolute ethanol volume fraction is 15% to 45%. In some embodiments, the anhydrous 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 liquid composition has a pH of 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, the edta.2na salt concentration is 0.5mM, the guanidine hydrochloride concentration is 100mM, the sodium chloride concentration is 50mM, and the 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 nuclear-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 use of the kit of the third aspect for extracting viral RNA from a sample containing sodium heparin. In some embodiments, there is provided the use of a kit of the third aspect for co-extraction of DNA and RNA nucleic acids on a sample comprising heparin sodium.
In a fifth aspect, the present disclosure provides a method for nucleic acid extraction from a sample, the method comprising lysing the sample, binding the nucleic acid to an adsorption column, washing the adsorption column, rinsing the adsorption column, and eluting the nucleic acid, wherein the liquid composition of the first aspect is used to lyse the sample and/or the liquid composition of the second aspect is used to wash the adsorption column.
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 the virus nucleic acid, has a better effect on removing the heparin sodium, and reduces the influence of the 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
As used herein, "buffer material" means a material capable of maintaining a solution pH environment and includes tris, 3- (N-morpholine) propanesulfonic acid (i.e., mops), sodium acetate, sodium citrate, sodium carbonate, and the like.
As used herein, "denaturant" refers to a denaturant having the action of cleaving viral particles, denaturing proteins, inhibiting RNase, providing a high-salt environment, and capable of promoting binding of viral DNA and/or RNA to an adsorption membrane, and includes guanidinium isothiocyanate, guanidinium hydrochloride, guanidinium thiocyanate, perchlorate, naI, KI, urea, and the like.
As used herein, "surfactant" means a substance having the action of solubilizing and denaturing proteins in a sample, and includes Triton X-100, tween-20, tween-80, NP-40, SDS, CHAPS, sodium deoxycholate, sodium cholate, betaine, PEG8000 and the like.
As used herein, "metal ion chelating agent" means a substance having an action of chelating a metal ion, and includes EDTA.2Na, EDTA.4Na, EDTA, sodium citrate, EGTA, HEDTA and the like.
Drawings
FIG. 1: extracting a QPCR result of PEDV nucleic acid in human plasma from different lysate;
FIG. 2: extracting a qPCR result of PRV nucleic acid in human plasma from different lysate;
FIG. 3: the QPCR result of the PEDV nucleic acid in the human plasma is extracted from the three lysis solutions in the example 2;
FIG. 4: the QPCR result of PEDV nucleic acid in human plasma is extracted by three washing solutions in example 3;
FIG. 5: the qPCR results of PEDV nucleic acid in human plasma were extracted from the two washes in example 4;
FIG. 6: the results of qPCR for PEDV nucleic acid in human plasma were extracted in two different combinations in example 5.
Detailed description of the preferred embodiment (examples)
The technical solutions of the present disclosure are further described in the following detailed description with reference to the drawings, but the following examples are only simple examples of the present disclosure and do not represent or limit the scope of the present disclosure, which is defined by the claims.
In the following examples, reagents and consumables used were obtained from conventional reagent manufacturers in the field unless otherwise specified; unless otherwise indicated, all experimental methods and technical means are those conventional in the art.
Example 1: extracting virus samples containing heparin sodium from different lysates and detecting
1. Simulated sample preparation
Taking 10 bottles of Kejingjing vaccine (containing porcine epidemic diarrhea virus PEDV vaccine) (probiology, kejingjing) and 10 bottles of Kejingning vaccine (containing pseudorabies virus PRV vaccine) (probiology, kejingning), respectively adding 10ml of Kejingjing special vaccine diluent and Kejingning special vaccine diluent, fully dissolving and uniformly mixing, and respectively diluting two dissolved vaccines by 100 times in a gradient manner by using PBS. And adding 2 mu l of the diluted kefulin vaccine and 2 mu l of the diluted kefulin vaccine into 296 mu l of the human heparin sodium plasma sample, and fully mixing the mixture to prepare a sample of the simulated nucleic acid extract. The sample preparation is carried out according to the proportion expansion by using the actually required sample amount in each experiment.
2. Simulating sample lysis
20 mul PK, 300 mul of the simulated sample and 500 mul of lysate with different components are sequentially added into three groups of 1.5ml centrifuge tubes respectively, and after the mixture is fully shaken and uniformly mixed, the mixture is incubated for 5min at room temperature.
Different lysates were prepared as per table 1:
table 1: different lysate components and contents
| Components | Lysate 1 | |
Lysate 3 |
| Tris (hydroxymethyl) aminomethane | 20mM | 20mM | 20mM |
| EDTA·2Na·2H 2 O | 5mM | 5mM | / |
| Guanidine isothiocyanate | 2M | 2M | 2M |
| Betaine | 0.1mM | / | / |
| Tween-20 | 1％ | 1％ | 1％ |
| TritonX-100 | 0.1％ | 0.1％ | 0.1％ |
| PEG8000 | 0.005％ | 0.005％ | / |
| |
32％ | 32％ | 32％ |
| pH | 7.3 | 7.3 | 7.3 |
3. Nucleic acid binding adsorption column
Transferring all the three incubated lysis samples into three adsorption columns, centrifuging at 12000rpm for 1min, and discarding the waste liquid.
4. Washing adsorption column
700. Mu.l of the washing solution was added to each of the three adsorption columns, and the mixture was centrifuged at 12000rpm for 30sec, and the waste liquid was discarded. Wherein the used washing liquid comprises the following components in percentage by weight: 20mM Tris, 10mM EDTA.2Na2H 2 O, 2M guanidine hydrochloride, 0.002% Tween-20, 40% absolute ethyl alcohol, and the pH value of the washing solution is 7.5 (the washing solution in the proportion is named as washing solution 1).
5. Rinsing adsorption column
To the washed adsorption column, 700. Mu.l of a rinsing solution was added, and the mixture was centrifuged at 12000rpm for 30sec to discard the waste liquid. Wherein the rinsing liquid comprises the following components in percentage by weight: 20mM Tris, 0.5mM EDTA.2Na. 2H 2 O, 100mM guanidine hydrochloride, 50mM sodium chloride, 80% absolute ethyl alcohol, and the pH value of a rinsing liquid is 8.0 (the rinsing liquid in the proportion is named as rinsing liquid 1).
6. Elution of cleavage products
Firstly, putting the adsorption column in the previous step into a centrifuge, centrifuging at 12000rpm for 2min, then transferring the adsorption column into a new 1.5mL centrifuge tube, adding 50 mul of eluent, standing for 1min, and centrifuging at 12000rpm for 1min to obtain three solutions, namely virus DNA/RNA solution extracted from the simulation sample. Wherein the components and contents of the eluent used are 10mM tris, nuclear-free Water and the pH of the eluent is 6.0.
7. Detecting the extracted product
Using HiScript II U + The One Step qRT-PCR Probe Kit (Novozam organism, cat # Q222-CN) Kit carries out qPCR detection on the extracted product.
PEDV and PRV viral genes were detected on the extracted product according to the reaction system of table 2 and the reaction procedure of table 3.
Table 2: qPCR reaction system
| RNase-freeddH 2 O | 9.4 |
| 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 | 30μl |
Table 3: qPCR reaction procedure
The sequences of the primer probes used for detecting the PEDV and PRV viral genes are shown in Table 4.
Table 4: PEDV and PRV virus gene detection primer probe sequence
| Sequence numbering | Sequence name | Sequence of |
| 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
The qPCR results are shown in fig. 1 (PEDV virus detection results) and fig. 2 (PRV virus detection results), wherein L1 represents the qPCR result of the product obtained by lysis of the mock sample by lysate 1, L2 represents the qPCR result of the product obtained by lysis of the mock sample by lysate 2, and L3 represents the qPCR result of the product obtained by lysis of the mock sample by lysate 3. FIG. 1 shows that three lysates with different formulas have equivalent extraction effect when used for extracting PEDV, and FIG. 2 shows that three lysates with different formulas have equivalent extraction effect when used for extracting PRV.
Example 2: the amplification efficiency of the extracted product can be effectively improved by reducing the ethanol content in the lysate
Preparing lysis solution 3, lysis solution 4 and lysis solution 5 according to table 5, respectively, performing viral nucleic acid lysis extraction on the simulated sample by using lysis solution 3, lysis solution 4 and lysis solution 5 to obtain three extracted products, performing qPCR viral gene detection on the three extracted products, and comparing the influence of the three lysis solutions on the extracted products.
Table 5: lysate 3, lysate 4 and lysate 5 components and contents
| Components | Lysate 3 | |
Lysate 5 |
| Tris (hydroxymethyl) aminomethane | 20mM | 20mM | 20mM |
| Guanidine isothiocyanate | 2M | 2M | 2M |
| Tween-20 | 1％ | 1％ | 1％ |
| TritonX-100 | 0.1％ | 0.1％ | 0.1 |
| Anhydrous ethanol | |||
| 32％ | 24％ | 40％ | |
| pH | 7.3 | 7.3 | 7.3 |
The preparation of the mock sample, the rinsing solution, the eluent and the qPCR reaction in this example were the same as those in example 1, and the washing solution used in this example had a composition and a ratio of 20mM Tris, 10mM EDTA.2Na. 2H2H2mM 2 O, 2M guanidine hydrochloride, 0.002% Tween-20, 57% absolute ethyl alcohol, and the pH value of the washing solution is 7.5 (the washing solution in the ratio is named as washing solution 2).
Analysis of results
The results of example 2 are shown in fig. 3, wherein L3, L4, and L5 represent the results of qPCR for PEDV gene detection using the extracted products obtained from lysate 3, lysate 4, and lysate 5, respectively. As can be seen from the attached figure 3, the effect of extracting PEDV virus by using the lysate 3 and the lysate 4 is equivalent, and the effect of extracting PEDV virus by using the lysate 5 is the worst, which shows that the ethanol content in the lysate is reduced, and the amplification efficiency of the PEDV extraction product can be effectively improved.
Example 3: influence of three different washing solutions on amplification efficiency of extracted product
Table 6: washing liquid 2, washing liquid 3 and washing liquid 4 components and contents
| | Washing solution | 2 | Washing solution 3 | |
| Tris (hydroxymethyl) aminomethane | 20mM | 20mM | 20mM | |
| EDTA·2Na·2H 2 O | 10mM | 10mM | 10mM | |
| Guanidine hydrochloride | 2M | 2M | 2M | |
| Tween-20 | 0.002％ | 0.002％ | 0.002％ | |
| Anhydrous ethanol | 57％ | 28％ | 17％ | |
| pH | 7.5 | 7.5 | 7.5 |
The preparation of the simulated sample, the rinsing solution, the eluent, and the qPCR reaction in this example were the same as those in example 1, and the lysis solution used in this example was lysis solution 5 in example 2.
Analysis of results
Example 3 the results are shown in figure 4, wherein W2, W3, W4 represent the results of qPCR for PEDV gene detection using extracts obtained from wash 2, wash 3, wash 4, respectively. As can be seen from FIG. 4, the amplification efficiency of PEDV extracted by using washing solutions 3 and 4 is equivalent, while the amplification efficiency of the product extracted by washing solution 2 is slightly poor, which indicates that the amplification efficiency of the PEDV extracted product can be effectively improved by reducing the ethanol content in the washing solution.
Example 4: effect of two different washing solutions on amplification efficiency of extracted product
Washing solution 1 and washing solution 2 are prepared according to example 1 and example 3 respectively, a simulation sample is extracted by using washing solution 1 and washing solution 2 respectively to obtain two extracted products, qPCR virus gene detection is carried out on the two extracted products, and the influence of the two washing solutions on the amplification efficiency of the extracted products is compared. The preparation of the simulated sample, the rinsing solution, the eluent and the qPCR reaction in this example are the same as those in example 1, and the lysis solution used in this example is lysis solution 5 in example 2.
Analysis of results
Example 4 the results are shown in FIG. 5, wherein W1 and W2 represent the qPCR results of PEDV gene detection using the extract obtained from Wash 1 and Wash 2, respectively. As can be seen from FIG. 5, the amplification efficiency of PEDV extracted by using washing solution 1 is significantly better than that of washing solution 2, indicating that reducing the ethanol content in the washing solution can effectively improve the quality of PEDV product.
Example 5: simultaneously, the ethanol content in the lysate and the washing liquid is reduced, so that the amplification efficiency of the extracted product can be effectively improved
And (2) performing nucleic acid extraction on the simulated biological sample by using a lysis solution 5 and a washing solution 2 (named as a combination 1), performing nucleic acid extraction on the simulated biological sample by using a lysis solution 4 and a washing solution 1 (named as a combination 2), performing qPCR (quantitative polymerase chain reaction) virus gene detection on two nucleic acid extraction products, and comparing the influence of the two combinations on the amplification efficiency of the extraction products. The preparation of the mock sample, the rinsing solution, the eluent, and the qPCR reaction in this example were the same as in example 1.
Analysis of results
The results of example 5 are shown in fig. 6, where beform stands for the PEDV gene detection qPCR results for the extracted products obtained using combination 1, and After stands for the PEDV gene detection qPCR results for the extracted products obtained using combination 2. It can be seen from fig. 6 that the amplification efficiency of PEDV extracted by using combination 2 is significantly better than that of combination 1, which indicates that the reduction of ethanol content in the lysate and the washing solution can effectively improve the amplification efficiency of PEDV product.
Claims (25)
1. A liquid composition comprises a buffer substance, a denaturant, a surfactant and absolute ethyl alcohol, wherein the volume fraction of the absolute ethyl alcohol is 20-35%.
2. The liquid composition of claim 1, wherein the volume fraction of the anhydrous ethanol is 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%.
3. The liquid composition of claim 1, wherein the buffer material is selected from at least one of tris, 3- (N-morpholine) propanesulfonic acid, sodium acetate, sodium citrate, sodium carbonate.
4. The liquid composition of claim 1, wherein the denaturant is at least one selected from the group consisting of guanidinium isothiocyanate, guanidinium hydrochloride, guanidinium thiocyanate, perchlorate, naI, KI, and urea.
5. The liquid composition of claim 1, wherein 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, and PEG 8000.
6. The liquid composition of claim 1, comprising tris, guanidinium isothiocyanate, tween-20, triton X-100, and absolute ethanol, wherein the volume fraction of absolute ethanol is 20-35%.
7. The liquid composition according to claim 6, wherein the concentration of tris is 20mM, the concentration of guanidinium isothiocyanate is 2M, the volume fraction of Tween-20 is 1%, the volume fraction of Triton X100 is 0.1%, and 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%.
8. The liquid composition according to claim 7, further comprising a metal ion chelating agent selected from at least one of EDTA-2 Na, EDTA-4 Na, EDTA, sodium citrate, EGTA, HEDTA, preferably the metal ion chelating agent is EDTA-2 Na, at a concentration of 5mM.
9. The liquid composition of claim 8, further comprising betaine at a concentration of 0.1mM.
10. The liquid composition of claim 9, further comprising PEG8000, wherein the volume fraction of the PEG8000 is 0.005%.
11. A liquid composition according to any one of claims 1 to 10 having a pH of from 7.0 to 7.5, preferably a pH of 7.3.
12. A liquid composition comprising a buffer substance, a metal ion chelating agent, a denaturant, a surfactant and anhydrous ethanol, wherein the volume fraction of the anhydrous ethanol is 15% to 45%.
13. The liquid composition of claim 12, wherein the volume fraction of absolute ethanol 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%.
14. The liquid composition of claim 12, wherein the buffer material is selected from at least one of tris, 3- (N-morpholine) propanesulfonic acid, sodium acetate, sodium citrate, sodium carbonate; the metal ion chelating agent is at least one selected from EDTA-2 Na, EDTA-4 Na, EDTA, sodium citrate, EGTA and HEDTA; the denaturant is at least one of guanidinium isothiocyanate, guanidinium hydrochloride, guanidinium thiocyanate, perchlorate, naI, KI and urea; the surfactant is at least one, at least two or at least three of Triton X-100, tween-20, tween-80, NP-40, SDS, CHAPS, sodium deoxycholate and sodium cholate.
15. The liquid composition according to claim 14, which comprises tris, EDTA-2 Na salt, guanidine hydrochloride, tween-20 and absolute ethanol, wherein the volume fraction of the absolute ethanol is 15% to 45%.
16. The liquid composition according to claim 15, wherein the concentration of tris is 20mM, the concentration of EDTA-2 Na salt is 10mM, the concentration of guanidine hydrochloride is 2M, the volume fraction of tween-20 is 0.002%, and the volume fraction of the absolute ethyl alcohol 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%.
17. A liquid composition according to any one of claims 12 to 16 having a pH of from 7.3 to 7.7, preferably a pH of 7.5.
18. A kit comprising the liquid composition according to any one of claims 1 to 11 and/or the kit comprising the liquid composition according to any one of claims 12 to 17.
19. The kit of claim 18, further comprising a liquid composition for rinsing the adsorption column, the liquid composition comprising tris, EDTA-2 Na salt, guanidine hydrochloride, sodium chloride, and ethanol.
20. The kit according to claim 19, wherein the concentration of tris is 20mM, the concentration of EDTA-2 Na salt is 0.5mM, the concentration of guanidine hydrochloride is 100mM, the concentration of sodium chloride is 50mM, the volume fraction of ethanol is 80%, and the pH of the liquid composition is 8.0.
21. The kit according to claim 18, further comprising a liquid composition for eluting nucleic acid, wherein the liquid composition comprises Tris (hydroxymethyl) aminomethane at a concentration of 10mM and Nuclear-free Water, and the liquid composition has a pH of 6.0.
22. Use of a kit according to any one of claims 18 to 21 for the extraction of viral RNA or for the co-extraction of DNA and RNA nucleic acids from a sample containing sodium heparin.
23. A method for nucleic acid extraction from a sample, the method comprising lysing the sample, binding the nucleic acid to an adsorption column, washing the adsorption column, rinsing the adsorption column, eluting the nucleic acid, wherein the sample is lysed using a liquid composition according to any one of claims 1 to 11 and/or the adsorption column is washed using a liquid composition according to any one of claims 12 to 17.
24. The method for extracting nucleic acid from a sample according to claim 23, wherein the sample is a plasma sample containing heparin sodium.
25. The method for extracting nucleic acid from a sample according to any one of claims 23 to 24, wherein the method is used for extracting viral RNA from a sample or for extracting DNA and RNA from a sample together.
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