CN102250886A - Extraction method of goose parvovirus DNA (deoxyribonucleic acid) - Google Patents
Extraction method of goose parvovirus DNA (deoxyribonucleic acid) Download PDFInfo
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- CN102250886A CN102250886A CN 201110223119 CN201110223119A CN102250886A CN 102250886 A CN102250886 A CN 102250886A CN 201110223119 CN201110223119 CN 201110223119 CN 201110223119 A CN201110223119 A CN 201110223119A CN 102250886 A CN102250886 A CN 102250886A
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Abstract
The invention relates to an extraction method of goose parvovirus DNA (deoxyribonucleic acid), belonging to the field of biotechnology. The extraction method comprises the following steps: adding equal-volume of chloroform to a goose embryo allantoic fluid containing goose parvovirus, vibrating at room temperature for 15-20 minutes, centrifuging at 3500 rpm for 10 minutes, sucking supernatant, then adding equal-volume of chloroform, vibrating, centrifuging, repeating three times, sucking the supernatant, adding the supernatant into an EP (electropolished) tube, boiling in boiling water for 3-5 minutes, centrifuging at 8000-10000 rpm for 5 minutes, and sucking the supernatant to obtain the goose parvovirus DNA. In the extraction method provided by the invention, only chloroform is used as the reagent, and the virus is split to the allantoic fluid and fat and protein large particles are removed by repeated freezing and thawing, boiling and centrifuging; and the extraction method is convenient for operation, has low cost and high efficiency and is convenient for laboratory operation.
Description
Technical field
The present invention relates to a kind of method of goose parvovirus DNA extraction, belong to biological technical field.
Background technology
Trichloromethane (Trichloromethane) has another name called chloroform, and molecular formula is [CHCl
3].For three hydrogen atoms in the methane molecule are replaced the compound that generates by chlorine.Be water white heavy liquid, highly volatile, flavor Xin Tianer has special aromatic odour.Miscible with ethanol, ether, benzene, sherwood oil, tetracol phenixin, dithiocarbonic anhydride and volatilization wet goods, be slightly soluble in water (25 ℃ time 1ml be dissolved in about 200ml water).It mainly act as: 1. trichloromethane is the organic synthesis raw material, is mainly used to produce fluorine Lyons (F-21, F-22, F-23).In addition, also be used for organic synthesis and narcotic; The solvent of fat, rubber, resin, oils, wax, phosphorus, iodine and bonding acryl; The extraction agent of penicillin, essential oil, alkaloid etc.; Measure inorganic phosphorus in the serum; Clean-out system; The sanitas of liver function test etc.It is the clean-out system of Cellphone Repairs personnel indispensability.2. chloroform mixes with tetracol phenixin and can be made into the fire prevention liquid that does not freeze.Also be used for the propelling charge of smoke substance, the fumigant of cereal and the reference liquid of base measuring temperature.Industrial products are added with small amount of ethanol usually, make the phosgene of generation and ethanol effect generate nontoxic diethyl carbonate.
Goose Parvovirus (Goose parvovirus infection) claims gosling plague (Gosling plague) again.It is a kind of acute infectious disease of day old chick goose.Be that (Goose parvovirus GPV) causes, this virus is spherical, and diameter is 20-22nm, Single-stranded DNA virus by goose parvovirus.The extraction that contains GPV viral DNA in the GPV goose embryo allantoic liquid adopts phenol and chloroform/primary isoamyl alcohol method and DNA extraction test kit to extract usually.Phenol and chloroform/primary isoamyl alcohol method is by several agents, mainly uses phenol/chloroform/primary isoamyl alcohol, Proteinase K, sodium-chlor, reaches the method that sodium lauryl sulphate reagent such as (SDS) carries out DNA extraction.The DNA extraction kit method promptly is to adopt commercially available battery of tests box to finish the extraction of DNA.Phenol and chloroform/required reagent of primary isoamyl alcohol method is many, the preparation trouble, and process is loaded down with trivial details.And test kit costs an arm and a leg, and does not have the small volume of reagent box, and operating process is strict, is inconvenient to use.
And present method is at first with toxic goose embryo allantoic liquid multigelation, and purpose is to make protein denaturation, and makes part contain the virocyte fragmentation.Utilizing DNA is polar compound, generally all water-soluble, is insoluble to organic solvents such as chloroform, ethanol.And protein can form protein gel with chloroform, and the lipid in the allantoic fluid also dissolves in chloroform simultaneously, and the vibration back forms suspension liquid, and through centrifugal, protein gel rests on water and chloroform is middle mutually, and DNA is positioned at the aqueous phase on upper strata.Due to illness poison is present in the cell of allantoic fluid, and the cleavage method of cell is a lot, as stain remover method, boiling water pyrolysis method, alkaline denaturation, organic solvent method and bacteriolyze enzyme process.The present invention is the pyrolysis method that adopts, and gets the water that contains DNA, carries out cracking by boiling pair cell, makes it discharge DNA.
Summary of the invention
Technical problem the objective of the invention is with a kind of fast, cheapness and the high-efficiency method goose parvovirus DNA that will be present in the goose embryo allantoic liquid extracts.
The technical scheme concrete steps are, contain the goose embryo allantoic liquid multigelation three times of goose parvovirus after, the trichloromethane of equivalent volumes ratio is joined in this allantoic fluid, vibrated under the room temperature 15～20 minutes, centrifugal 10 minutes of 3500rpm draws supernatant, adds the trichloromethane of equivalent volumes again, vibration, centrifugal, three times so repeatedly, draw supernatant, in the EP pipe of packing into, boiling water boiled 3～5 minutes, centrifugal 5 minutes of 8000～10000rpm.The absorption supernatant promptly gets and contains the goose parvovirus dna solution.
Beneficial effect
Agents useful for same of the present invention only is that trichloromethane is a kind of, by vibration repeatedly, removes fat and protein macrobead in the allantoic fluid, by multigelation, boil, make lysis, virus enters in the allantoic fluid, and its security is good, is convenient to operation, with low cost, the efficient height is convenient to laboratory operation.
Advantage of the present invention is:
1, security is good: agents useful for same only is a trichloromethane in the method for the present invention, removes fat, protein and cell debris in the allantoic fluid by method of extraction, and virus is retained in the supernatant liquor.
2, with low cost: used starting material wide material sources in the method for the present invention, with low cost, equipment used is simple, energy consumption is extremely low, to take cost be traditional phenol and chloroform/below 1/3 of primary isoamyl alcohol method, be below 1/10 of DNA extraction test kit (import).
3, easy and simple to handle: operating process of the present invention is extremely easy, and agents useful for same is only a kind of, only needs to carry out in common different size centrifuge tube or Glass Containers.
4, efficient height: method of the present invention can extract the goose parvovirus DNA that breeds in the goose embryo allantoic liquid effectively, the extraction yield height.And traditional method and kit method are in use, because of reagent preparation, preservation and operational issue, tend to occur the failure phenomenon.
Description of drawings
The extracting method schema of Fig. 1 goose parvovirus DNA
Fig. 2 contains the goose embryo allantoic liquid PCR measurement result of goose parvovirus
1, contains the GPV venom; 2, contain the GPV venom after the dilution; 3, with a collection of venom, boiled 3 minutes without the aforesaid method processing; 4, identical venom with 3, the time of just boiling doubles; 5, positive control (dna profiling of carrying with test kit); 6, DNA marker (DL 2000)
Fig. 3 contains different layers solution PCR measurement result after the goose parvovirus goose embryo allantoic liquid sucrose density gradient centrifugation
The 1-8 road is the 1-8 floor solution of sucking-off from top to bottom from No. 1 centrifuge tube; The 10-17 road is the 1-8 floor solution of sucking-off from top to bottom from No. 2 centrifuge tubes; 9,18 is DNA marker (DL 2000)
Fig. 4 electron microscopic observation result.
Embodiment
The extraction of goose parvovirus DNA in example 1, the goose embryo allantoic liquid
Goose embryo allantoic liquid (the isolation identification Liu Yu Zhuo of goose parvovirus LH strain that will contain goose parvovirus LH strain; Li Yin; Wei Xuetao; Zhang Jingfeng Jiangsu agricultural journal 2009-10-31) put-20 ℃ freezing, treat to freeze fully the taking-up of good back and put the room temperature thawing, so repeatedly after three times, get trichloromethane (analytical pure AR, chemical reagent company limited is rather tried in Nanjing, relative molecular mass 119.38 meets: GB/682-89, and technical requirements: trichloromethane (CHCL3) content is no less than 99.0%, ethanol (C2H5OH) content (stablizer) 0.3%-1.0%, density (g/ml) 1.471-1.484) joins in the above-mentioned allantoic fluid of equivalent, at room temperature vibrated 20 minutes, centrifugal 10 minutes of 3500rpm, draw supernatant, add isopyknic trichloromethane again, vibration, centrifugal, so repeatedly after three times, draw supernatant, pack in the 1.5ml EP pipe, boiled 3 minutes, centrifugal 5 minutes of 10000rpm.Draw supernatant and carry out PCR mensuration, the results are shown in Figure 2.
As shown in Figure 2: 1st, 2 swimming lanes are the allantoic fluid of handling with present method that contains GPV, and wherein the 1st swimming lane is toxic allantoic fluid stoste, and the 2nd swimming lane is the allantoic fluid of 10 times of dilutions.Bright electrophoretic band appears in the 1st swimming lane, and GPV viral level height is described, the DNA of extraction is more; The 2nd swimming lane dilutes because of venom, though band appears, and lighter.Article two, the 44bp stripe size conforms to expected results, through the purpose band being reclaimed, expresses and transforms, checking order, reaches 99% with the GPV genes involved sequence homology of having delivered on the GENBANK, shows and extracted GPV nucleic acid from the venom sample.And the 3rd, 4 swimming lanes all do not carry out the processing in early stage with the inventive method, only carry out boiling of different time, the purpose band all do not occur through PCR.The PCR that 5 swimming lanes carry out for the dna profiling that extracts with test kit is as positive control.
Example 2,Concentrate and contain GPV goose embryo allantoic liquid different layers solution PCR product electrophoresis result after sucrose density gradient centrifugation
Goose embryo allantoic liquid (the isolation identification Liu Yu Zhuo of goose parvovirus LH strain that will contain goose parvovirus LH strain; Li Yin; Wei Xuetao; Zhang Jingfeng Jiangsu agricultural journal 2009-10-31),, trichloromethane freezing by the inventive method handled, draw supernatant after PEG concentrates, be added on the solution of different sucrose density gradients, centrifugal 4 hours of 38000rpm, the layering draw solution is preserved, and draws each layer solution and packs in the EP centrifuge tube, boiling water boiled 5 minutes, centrifugal 5 minutes of 8000rpm.Draw supernatant and carry out PCR mensuration, the results are shown in Figure 3.Get the PCR electrophoresis and slice layer solution occurs,, can see the GPV particle, see Fig. 4 by electron microscopic observation after the negative staining.
As shown in Figure 3: the 1st～8 road is 1～8 a floor sucrose solution of sucking-off from up to down from No. 1 centrifuge tube; 10～17 roads are 1～8 floor sucrose solution of sucking-off from up to down from No. 2 centrifuge tubes, and the 9th, 18 two swimming lane is respectively DNA marker (DL 2000).PCR product electrophorogram shows that electrophoretic band all appears in the 6th, 7,8 three swimming lane and 15,16,17 3 swimming lanes, conforms to purpose band 440bp size, shows and contains GPV nucleic acid in the venom.Explanation all contains GPV in back three gradients in two centrifuge tubes, and the purpose band all do not occur through PCR in other gradient, illustrates and does not contain GPV.
As shown in Figure 4: behind the ultracentrifugation, get the PCR reaction product electrophoresis layer solution that is positive, through the phospho-wolframic acid negative staining, electron microscopic observation is seen diameter and is the spherical virus particle about 20nm, conform to GPV size and shape, illustrate and contain GPV virus in this layer, and the PCR reaction product electrophoresis layer solution that is negative, negative staining is observed, do not see virus like particle, conform to the PCR test-results.
Claims (1)
1. the method for goose parvovirus DNA extraction comprises:
After containing the goose embryo allantoic liquid multigelation three times of goose parvovirus, the trichloromethane of equivalent volumes ratio is joined in the goose embryo allantoic liquid that contains goose parvovirus, vibrated under the room temperature 15～20 minutes, centrifugal 10 minutes of 3500rpm draws supernatant; The trichloromethane that adds equivalent volumes again, supernatant is drawn in vibration, centrifugal; Three times so repeatedly, supernatant is packed in the EP pipe, and boiling water boiled 3～5 minutes, and centrifugal 5 minutes of 8000～10000rpm draws supernatant and promptly gets the DNA that contains goose parvovirus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106893732A (en) * | 2017-04-18 | 2017-06-27 | 扬州大学 | A kind of rescue method of Goose Parvovirus clone |
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Non-Patent Citations (3)
| Title |
|---|
| 《医学研究生学报》 20010831 周宗安，等 不同传染性腔上囊病病毒株结构蛋白表达差异性研究 285-288 1 第14卷, 第4期 * |
| 《江苏农业学报》 20091231 刘宇卓，等. 鹅细小病毒 LH株的分离鉴定 1091-1094 1 第25卷, 第5期 * |
| 《浙江农业学报浙江农业学报》 20091231 魏战勇，等. 精液中猪细小病毒DNA三种提取方法效果比较 455-458 1 第21卷, 第5期 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106893732A (en) * | 2017-04-18 | 2017-06-27 | 扬州大学 | A kind of rescue method of Goose Parvovirus clone |
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Application publication date: 20111123 |