CN111235143A - Extraction method of virus nucleic acid - Google Patents
Extraction method of virus nucleic acid Download PDFInfo
- Publication number
- CN111235143A CN111235143A CN202010124653.6A CN202010124653A CN111235143A CN 111235143 A CN111235143 A CN 111235143A CN 202010124653 A CN202010124653 A CN 202010124653A CN 111235143 A CN111235143 A CN 111235143A
- Authority
- CN
- China
- Prior art keywords
- dna
- tissue
- nucleic acid
- adsorption column
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 34
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 33
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 33
- 241000700605 Viruses Species 0.000 title claims abstract description 22
- 238000000605 extraction Methods 0.000 title description 3
- 108020004414 DNA Proteins 0.000 claims abstract description 43
- 238000001179 sorption measurement Methods 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 23
- 239000006228 supernatant Substances 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 12
- 238000000265 homogenisation Methods 0.000 claims abstract description 8
- 239000003480 eluent Substances 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 102000053602 DNA Human genes 0.000 claims abstract description 4
- 108091092584 GDNA Proteins 0.000 claims abstract description 4
- 210000002216 heart Anatomy 0.000 claims abstract description 4
- 210000004185 liver Anatomy 0.000 claims abstract description 4
- 210000004072 lung Anatomy 0.000 claims abstract description 4
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 4
- 229920002477 rna polymer Polymers 0.000 claims abstract description 4
- 238000012163 sequencing technique Methods 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 230000003612 virological effect Effects 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 230000009089 cytolysis Effects 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 3
- 150000002632 lipids Chemical class 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—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 chromatography, e.g. electrophoresis, ion-exchange, reverse phase
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting virus nucleic acid, which comprises the following steps: preparing samples, namely preparing animal tissues infected by viruses, tissue samples including heart, liver, lung and other tissues, and removing background nucleic acids such as gDNA (deoxyribonucleic acid) and rRNA (ribonucleic acid) of a host before the nucleic acid interference sequencing of the host is carried out; extracting tissue, namely putting appropriate animal tissue into a beaker, adding 3 times of physiological saline for homogenization, and centrifuging at 10000rpm for 2min after sufficient homogenization to remove residual tissue; and (3) splitting virus, taking appropriate supernatant into a new clean centrifugal tube, and adding appropriate DNA extracting solution into the supernatant. The invention cracks cells by mixing the DNA extracting solution and the supernatant, and finally obtains the DNA solution by washing through a washing solution and an eluent multi-adsorption column.
Description
Technical Field
The invention relates to the technical field of viral nucleic acid, in particular to a method for extracting viral nucleic acid.
Background
A virus is a noncellular organism that is small in size, simple in structure, contains only one nucleic acid (DNA or RNA), and must be parasitic in living cells and proliferated in a replicative manner. The virus is a non-cell life form, which is composed of a long nucleic acid chain and a protein shell, and has no own metabolic mechanism and no enzyme system. Therefore, the virus leaves the host cell and becomes a chemical substance which does not have any vital activity and can not independently propagate. Its ability to replicate, transcribe, and translate is performed in the host cell, and when it enters the host cell, it can use the materials and energy in the cell to perform life activities, generating a new generation of virus as it does according to the genetic information contained in its own nucleic acid.
Since viruses are various in types and are easy to mutate, nucleic acid in viruses needs to be extracted and analyzed every time a new virus is developed, and the conventional extraction method has various steps and is inconvenient to operate.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for extracting viral nucleic acid.
The invention provides a method for extracting viral nucleic acid, which comprises the following steps:
s1: preparing samples, namely preparing animal tissues infected by viruses, tissue samples including heart, liver, lung and other tissues, and removing background nucleic acids such as gDNA (deoxyribonucleic acid) and rRNA (ribonucleic acid) of a host before the nucleic acid interference sequencing of the host is carried out;
s2: extracting tissue, namely putting appropriate animal tissue into a beaker, adding 3 times of physiological saline for homogenization, and centrifuging at 10000rpm for 2min after sufficient homogenization to remove residual tissue;
s3: splitting virus, taking appropriate supernatant into a new clean centrifuge tube, adding appropriate DNA extract into the supernatant, reversing, mixing, and standing at room temperature for 5-10min to split virus;
s4: washing, namely, putting the DNA adsorption column into a collecting pipe, transferring all cracked liquid into the DNA adsorption column, centrifuging for 1min at 10000rpm, dumping filtrate, putting the DNA adsorption column back into the collecting pipe, adding proper washing liquid into the DNA adsorption column, centrifuging for 1min at 10000rpm, dumping filtrate in the collecting pipe, putting the DNA adsorption column back into the collecting pipe, and centrifuging for 3min at 10,000 rpm;
s5: DNA solution, the DNA adsorption column is arranged in a new clean centrifuge tube, proper eluent is added to the center of the adsorption column, standing is carried out for 1-2min, and centrifugation is carried out for 1min at 10,000rpm, thus obtaining the DNA solution;
s6; for storage, the DNA solution should be stored at-20 ℃.
Preferably, in S3, the supernatant and the DNA extract are mixed to cause cell disruption, thereby releasing the nucleic acid from the cell.
Preferably, in S3, the released nucleic acid is specifically adsorbed on a specific carrier having a strong affinity and adsorbability for nucleic acid, and other biochemical components are not substantially adsorbed into proteins, polysaccharides and lipids, and thus are spun out of the column during centrifugation.
Preferably, in S5, the washing solution is added with absolute ethanol before the first use, and the cover is closed immediately after the first use to prevent ethanol volatilization.
Preferably, in S3, the tissue is cut as much as possible so as not to affect the lysis effect, and the tissue may be sticky after complete lysis.
The beneficial effects of the invention are as follows:
the method for extracting the virus nucleic acid cracks cells by mixing the DNA extracting solution and the supernatant, and finally obtains the DNA solution by cleaning through a washing solution and an eluent multi-adsorption column.
Drawings
FIG. 1 is a flow chart of a method for extracting viral nucleic acid according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to fig. 1, a method for extracting viral nucleic acid includes the following steps:
s1: preparing samples, namely preparing animal tissues infected by viruses, tissue samples including heart, liver, lung and other tissues, and removing background nucleic acids such as gDNA (deoxyribonucleic acid) and rRNA (ribonucleic acid) of a host before the nucleic acid interference sequencing of the host is carried out;
s2: extracting tissue, namely putting appropriate animal tissue into a beaker, adding 3 times of physiological saline for homogenization, and centrifuging at 10000rpm for 2min after sufficient homogenization to remove residual tissue;
s3: splitting virus, taking appropriate supernatant into a new clean centrifuge tube, adding appropriate DNA extract into the supernatant, reversing, mixing, and standing at room temperature for 5-10min to split virus;
s4: washing, namely, putting the DNA adsorption column into a collecting pipe, transferring all cracked liquid into the DNA adsorption column, centrifuging for 1min at 10000rpm, dumping filtrate, putting the DNA adsorption column back into the collecting pipe, adding proper washing liquid into the DNA adsorption column, centrifuging for 1min at 10000rpm, dumping filtrate in the collecting pipe, putting the DNA adsorption column back into the collecting pipe, and centrifuging for 3min at 10,000 rpm;
s5: DNA solution, the DNA adsorption column is arranged in a new clean centrifuge tube, proper eluent is added to the center of the adsorption column, standing is carried out for 1-2min, and centrifugation is carried out for 1min at 10,000rpm, thus obtaining the DNA solution;
s6; for storage, the DNA solution should be stored at-20 ℃.
In the invention, in S3, the supernatant and the DNA extracting solution are mixed to promote cell disruption and release nucleic acid in the cell, in S3, the released nucleic acid is specifically adsorbed on a specific carrier, the carrier only has strong affinity and adsorption force on the nucleic acid, and other biochemical components enter protein, polysaccharide and lipid and are not adsorbed basically, so that the nucleic acid is thrown out of a column during centrifugation, in S5, before the washing solution is used for the first time, absolute ethyl alcohol is firstly added, and a cover is immediately covered after the washing solution is used to prevent the volatilization of the ethyl alcohol, in S3, tissues are cut as much as possible to avoid influencing the effect of lysis, and the nucleic acid can be sticky after being completely lysed.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A method for extracting viral nucleic acid, comprising the steps of:
s1: preparing samples, namely preparing animal tissues infected by viruses, tissue samples including heart, liver, lung and other tissues, and removing background nucleic acids such as gDNA (deoxyribonucleic acid) and rRNA (ribonucleic acid) of a host before the nucleic acid interference sequencing of the host is carried out;
s2: extracting tissue, namely putting appropriate animal tissue into a beaker, adding 3 times of physiological saline for homogenization, and centrifuging at 10000rpm for 2min after sufficient homogenization to remove residual tissue;
s3: splitting virus, taking appropriate supernatant into a new clean centrifuge tube, adding appropriate DNA extract into the supernatant, reversing, mixing, and standing at room temperature for 5-10min to split virus;
s4: washing, namely, putting the DNA adsorption column into a collecting pipe, transferring all cracked liquid into the DNA adsorption column, centrifuging for 1min at 10000rpm, dumping filtrate, putting the DNA adsorption column back into the collecting pipe, adding proper washing liquid into the DNA adsorption column, centrifuging for 1min at 10000rpm, dumping filtrate in the collecting pipe, putting the DNA adsorption column back into the collecting pipe, and centrifuging for 3min at 10,000 rpm;
s5: DNA solution, the DNA adsorption column is arranged in a new clean centrifuge tube, proper eluent is added to the center of the adsorption column, standing is carried out for 1-2min, and centrifugation is carried out for 1min at 10,000rpm, thus obtaining the DNA solution;
s6; for storage, the DNA solution should be stored at-20 ℃.
2. The method according to claim 1, wherein in S3, the supernatant and the DNA extract are mixed to break the cells and release the nucleic acids from the cells.
3. The method of claim 1, wherein the released nucleic acid is intentionally adsorbed onto a specific carrier having a strong affinity and adsorbability for nucleic acid, and other biochemical components such as proteins, polysaccharides and lipids are not substantially adsorbed, and thus are spun off the column during centrifugation in S3.
4. The method of claim 1, wherein in step S5, the washing solution is first added with absolute ethanol before first use, and then covered with a cover immediately after use to prevent ethanol volatilization.
5. The method according to claim 1, wherein in S3, the tissue is minced as much as possible so as not to affect the effect of lysis, and the tissue may be viscous after complete lysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010124653.6A CN111235143A (en) | 2020-02-27 | 2020-02-27 | Extraction method of virus nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010124653.6A CN111235143A (en) | 2020-02-27 | 2020-02-27 | Extraction method of virus nucleic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111235143A true CN111235143A (en) | 2020-06-05 |
Family
ID=70871539
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010124653.6A Pending CN111235143A (en) | 2020-02-27 | 2020-02-27 | Extraction method of virus nucleic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111235143A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114250222A (en) * | 2021-12-08 | 2022-03-29 | 苏州博腾生物制药有限公司 | Method for extracting AAV DNA from animal tissue |
| CN114606224A (en) * | 2022-03-24 | 2022-06-10 | 华南理工大学 | Method for extracting virus nucleic acid from animal tissue |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862305A (en) * | 2015-06-17 | 2015-08-26 | 河南省农业科学院畜牧兽医研究所 | Animal tissue genomic DNA and RNA rapid extraction kit, extraction method and application |
| CN104946624A (en) * | 2015-04-23 | 2015-09-30 | 中国水产科学研究院南海水产研究所 | Preparation method of total viral nucleic acid of shellfish |
| CN109385418A (en) * | 2017-08-03 | 2019-02-26 | 杭州优思达生物技术有限公司 | A kind of method extracted for virus in animal specimen/bacterial nucleic acid and reagent |
-
2020
- 2020-02-27 CN CN202010124653.6A patent/CN111235143A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104946624A (en) * | 2015-04-23 | 2015-09-30 | 中国水产科学研究院南海水产研究所 | Preparation method of total viral nucleic acid of shellfish |
| CN104862305A (en) * | 2015-06-17 | 2015-08-26 | 河南省农业科学院畜牧兽医研究所 | Animal tissue genomic DNA and RNA rapid extraction kit, extraction method and application |
| CN109385418A (en) * | 2017-08-03 | 2019-02-26 | 杭州优思达生物技术有限公司 | A kind of method extracted for virus in animal specimen/bacterial nucleic acid and reagent |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114250222A (en) * | 2021-12-08 | 2022-03-29 | 苏州博腾生物制药有限公司 | Method for extracting AAV DNA from animal tissue |
| CN114606224A (en) * | 2022-03-24 | 2022-06-10 | 华南理工大学 | Method for extracting virus nucleic acid from animal tissue |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bolduc et al. | Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs | |
| US20220275441A1 (en) | Barcode-free single vesicle multiplexed protein and rna analysis | |
| JP5127838B2 (en) | Methods and materials for stimulated release of biological samples | |
| CN111235143A (en) | Extraction method of virus nucleic acid | |
| JP3928973B2 (en) | Method, apparatus and reagent for isolating cellular components | |
| Singh et al. | SARS-CoV-2: emergence of new variants and effectiveness of vaccines | |
| JPH0515373A (en) | Method for extracting and purifying human genome dna | |
| CN103097532A (en) | Method for isolating a target nucleic acid including small target nucleic acids with high yield | |
| JP2002507121A (en) | RNA isolation method | |
| Palinauskas et al. | Laser microdissection microscopy and single cell PCR of avian hemosporidians | |
| Paim et al. | Characterization of the viral genomes present in commercial batches of horse serum obtained by high-throughput sequencing | |
| Van Thiel et al. | Can hepatitis B core antibody positive livers be used safely for transplantation: hepatitis B virus detection in the liver of individuals who are hepatitis B core antibody positive | |
| Dennin | DNA of free and complexed origin in human plasma: concentration and length distribution. | |
| JP6704563B2 (en) | Chaotrope and method for extracting genomic DNA using the chaotrope | |
| EP3587590A1 (en) | Nucleic acid depletion and detection | |
| CN110452874B (en) | Method for obtaining neuron | |
| CN111568928A (en) | Application of MSC (mesenchymal stem cell) in regulating and controlling gene expression of effector CD8+ T cells | |
| Vilhardt et al. | Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland | |
| CN114836540A (en) | Kit for detecting BCR/-ABL1 fusion gene and use method thereof | |
| CN111334867A (en) | Method for constructing virus nucleic acid library | |
| JPWO2018061877A1 (en) | Method for extracting nucleic acid and kit used therefor | |
| Strauss | Preparation of genomic DNA from mammalian tissue | |
| CN111529551A (en) | Application of MSC (mesenchymal stem cell) in adjusting number of plasma cells | |
| CN110951724A (en) | Method for rapidly extracting nucleic acid DNA and/or RNA by Trizol | |
| CN116162125A (en) | Method for separating and extracting nucleic acid binding protein in solid tissue cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |
|
| RJ01 | Rejection of invention patent application after publication |