CN102703436A - Method for extracting DNA of citrus canker by water bath precipitation - Google Patents
Method for extracting DNA of citrus canker by water bath precipitation Download PDFInfo
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- CN102703436A CN102703436A CN2012101969691A CN201210196969A CN102703436A CN 102703436 A CN102703436 A CN 102703436A CN 2012101969691 A CN2012101969691 A CN 2012101969691A CN 201210196969 A CN201210196969 A CN 201210196969A CN 102703436 A CN102703436 A CN 102703436A
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Abstract
The invention discloses a method for extracting DNA (Deoxyribonucleic Acid) of citrus canker by water bath precipitation. The method comprises the following steps: adding a washing buffer into a fresh thallus for washing; carrying out water bath by using a crashing buffer; extracting the buffer and a mixed solution of saturated phenol, chloroform and isoamyl alcohol; washing and precipitating ethanol; drying and adding sterilizing ddH2O for dissolving and precipitating; and preserving the precipitation at the temperature of -20 DEG C. According to the method disclosed by the invention, a template DNA of the citrus canker can be extracted quickly and effectively and the simpleness and quickness in operation are realized. The method dispenses with a special instrument, and thus the experiment cost is relatively-low. A DNA sample prepared by the method disclosed by the invention can meet the requirement of molecular diagnosis on the citrus canker by a PCR (Polymerase Chain Reaction) method can be met. Therefore, the invention constructs a method for quickly extracting the template DNA of the citrus canker by using a water bath method. The method provides a base and technical reserve for early-stage diagnosis and predicts detection applying the PCR technology.
Description
Technical field
The present invention relates to the DNA extraction method, the DNA water-bath deposition process for extracting of especially a kind of c itrus canker germ.
Background technology
Citrus bacterial canker disease be by carpetweed Xanthomonas campestris oranges and tangerines cause a disease mutation (
Xanthomonas axonopodisPv.
Citri) a kind of domestic and international quarantine venereal disease evil that causes, have a strong impact on oranges and tangerines safety in production and foreign trade, be put into " The People's Republic of China enter the territory Plant Quarantine property harmful organism register " that announced in 2007.
This disease can be through oranges and tangerines material long-distance communications such as germ-carrying seedling, scion and fruits, and snow mandarin orange, sweet orange, navel orange and four seasons article speciogenesis such as shaddock are comparatively serious.The germ that causes this disease survives the winter in the scab of leaf, branch and fruit, overflows from sick portion when spring in next year condition is suitable, borrows wind and rain, entomochory, invades through host's pore, hole skin and wound.Make the morbidity of blade, the tender tip and fruit, at present both at home and abroad except that excavate disease set burn, still do not have better elimination way.
The quarantine great majority of citrus bacterial canker disease are diagnosed as the master with the field at present; Can judge with naked eyes fully in the ordinary course of things; But unclear symptoms shows or does not manifest the relatively difficulty of plant judgement of carrying disease germs of symptom; Be prone to obscure with other diseases, simultaneously because kind and the mutation of Xanthomonas campestris are various, very numerous and diverse and difficult to the biochemical identification of citrus bacterial canker disease.At present the c itrus canker germ detection method used always of people comprises pathogenic, method based on use of bacteriophages for detection hiological and serological detection method etc.Along with development of molecular biology, begin to be applied to the detection of different diseases based on the molecule marker of DNA, because this method directly occurs with the form of DNA, can be more accurate than other field diagnosis.Therefore, set up a kind of method of c itrus canker germ DNA extraction, can not only be used for the Molecular Identification of c itrus canker germ, can also be used for the specific amplified of c itrus canker germ gene, for its functional genome research lays the foundation.
Summary of the invention
The objective of the invention is to solve the deficiency of above-mentioned prior art, the method for a kind of DNA that can fast extracting c itrus canker germ is provided,, thereby better foundation is provided for the prevention and control of citrus bacterial canker disease with the species diversity of overall understanding c itrus canker germ.
The technical solution adopted for the present invention to solve the technical problems is to carry out according to the following steps:
(a) new fresh thalli is added the lavation buffer solution thorough mixing, make the bacterial cell deposition, abandon supernatant;
(b) add lysis buffer in the deposition that makes toward step (a), vibration suspends, and in 65 ℃ of water-baths, complete reaction;
(c) extraction buffer of 65 ℃ of preheatings of adding, thorough mixing adds the saturated phenol of equal-volume, chloroform, the extracting of primary isoamyl alcohol mixing solutions, and the centrifugal albumen precipitation that makes sex change is got supernatant;
(d) be to add absolute ethyl alcohol in the supernatant that makes toward step (c) of the ratio of 1:2 according to volume ratio; After fully reaction is treated in 20 ℃ of placements of ﹣; High speed centrifugation to deposition is removed supernatant fully again, and adding volumetric concentration is the washing with alcohol deposition of 70 %, dries the back and adds sterilization ddH
2The O dissolution precipitation, 20 ℃ of preservations of ﹣ are subsequent use.
In the step (a), add lavation buffer solution 1 mL in per 0.12 g sample; Said lavation buffer solution consists of: 50 mmol/L Tris, i.e. and Tutofusin tris, pH 7.7; 25 mmol/L EDTA, i.e. YD 30s; 0.1 % PVP, i.e. Vinylpyrrolidone polymer.
In the step (b), add lysis buffer 50 μ L in per 0.12 g sample; Said lysis buffer consists of: 50 mmol/L Tris, and pH 8.0; 25 mmol/L EDTA; 3.0 % SDS, i.e. sodium lauryl sulphate; 1.2 % PVP.Water-bath time 5-10 min.
In the step (c), add extraction buffer 400 μ L in per 0.12 g sample; Said extraction buffer consists of: 50 mmol/L Tris, and pH 8.0; 25 mmol/L EDTA; 3.0 % SDS; 1.2 % PVP.
In the step (c), saturated phenol: chloroform: the primary isoamyl alcohol volume ratio is 25:24:1.
Effect of the present invention is that the present invention uses immersion method can fast and effeciently extract the template DNA of c itrus canker germ, and is simple and direct quick.This law does not need special instrument in addition, and experimental cost is relatively low.Use the DNA sample of the inventive method preparation can satisfy PCR method carries out molecular diagnosis to the c itrus canker germ needs.Therefore the present invention has set up the method with the template DNA of the quick extracting c itrus canker of immersion method germ, and detection provides basis and tachnical storage with prevision for Using P CR technology is carried out early diagnosis.
Description of drawings
Fig. 1: the agarose gel electrophoresis figure of c itrus canker germ DNA.
Among the figure, the dna molecular amount standard (DL 2000 DNA ladder) that Marker buys from biotech firm, 1,2 represent parallel appearance respectively, and numeral is the size of DNA, and unit is bp.
Fig. 2: the agarose gel electrophoresis figure of the PCR product of pcr amplification c itrus canker germ DNA.
Among the figure, the dna molecular amount standard (100bp DNA ladder) that Marker buys from biotech firm, 1,2 represent the parallel appearance of PCR product of c itrus canker germ DNA respectively, and numeral is the size of DNA, and unit is bp.
Embodiment
Employed TP is ordinary method like no specified otherwise among the following embodiment; Employed material, reagent etc. like no specified otherwise, are the reagent and the material that can obtain from commercial sources.
Embodiment 1: use the extraction of method of the present invention to c itrus canker germ DNA:
1, take by weighing the new fresh thalli of 0.12 g and add in the 1.5 mL centrifuge tubes, add lavation buffer solution 1 mL, vibration 30 s on mixing tank, centrifugal 2 min of 6,000 rpm make the bacterial cell deposition then, abandon supernatant.
Said lavation buffer solution consists of: 50 mmol/L Tris, pH 7.7,25 mmol/L EDTA, 0.1 % PVP.
2, in washed sample, add lysis buffer 50 μ L, vibration suspends, and in 65 ℃ of water-baths, handles 5-10 min, complete reaction.
Said lysis buffer consists of: 50 mmol/L Tris, pH 8.0,25 mmol/L EDTA, 3.0 % SDS, 1.2 % PVP.
3, the extraction buffer that adds 65 ℃ of preheatings of 400 μ L, the abundant mixing that vibrates, add the saturated phenol of equal-volume: chloroform: the extracting of primary isoamyl alcohol (25:24:1) solution, centrifugal 5 min of 10,000 rpm make the albumen precipitation of sex change, get supernatant.
Said extraction buffer consists of: 50 mmol/L Tris, pH 8.0,1 mmol/L EDTA, 0.3% mol/L NaCl, 0.1 % PVP.
4, get in addition in supernatant 400 μ L and the 1.5 mL centrifuge tubes, and add the absolute ethyl alcohol of 800 μ L precoolings, put 20 ℃ of refrigerator and cooled of ﹣ and freeze 30 min-60 min and impel deposition to form; The centrifugal 10 min deposit D NA of 12,000 rpm, and abandoning supernatant; With 1 mL, 70 % (V/V) washing with alcohol deposition, discard ethanol then as far as possible, dry naturally, make the ethanol volatilization fully, but can not make deposition too dry, otherwise be difficult to dissolving; Add 30 μ L sterilization ddH
2The O dissolution precipitation, 20 ℃ of preservations of ﹣ are subsequent use.
5, the agarose gel electrophoresis of dna fragmentation size detects
Get of the agarose gel electrophoresis detection of 5 μ L DNA nucleic acid with 1.2 %; With the detection of dyeing of 2 μ L nucleic acid dye Goldview I; Used dna molecular amount standard (Marker) is the DL 2000 DNA ladder of the biochemical ltd of sky, Beijing root; Used electrophoretic buffer is 1 * TAE electrophoretic buffer, with voltage prerunning 5 min of 120 V, and 100 V voltage prerunnings, 50 min then.Electrophoresis finishes later gel in the detection of taking pictures of the Gel Doc of U.S. BIORAD company 2000 gel imaging systems, and the result sees Fig. 1.
1 * TAE electrophoretic buffer wherein: 40 mmol/L Tris, 20 mmol/L acetate, 2 mmol/L EDTA; The sepharose of said 1.2 %: 0.36 g agarose adds 30 mL, 1 * TAE heating for dissolving, the 2 μ L nucleic acid dye Goldview I that add later on to be cooled.
6, the pcr amplification of the template DNA of c itrus canker germ and product detect
(1) as amplimer, the purpose fragment length is 373 bp to the Auele Specific Primer of selecting the c itrus canker germ to P 1 (5 ' TGCGGGCGTCCTCACAAAT 3 ') and P 2 (5 ' C CCGCCTTAGCCTACACGAC 3 ').
25 mL PCR reaction system: ddH
2O 18.85 μ L, 2.5 μ L, 10 * Buffer contains Mg
2+, 10 mM dNTP, 0.5 μ L, 1.0 μ L upstream primer P1 (10 pmole/10 μ L), 1.0 μ L downstream primer P3 (10 pmole/10 μ L), 0.15 μ L Taq archaeal dna polymerase (5 U/ μ L); The PCR circulation of primer amplification is: 94 ℃ of 4 min, 94 ℃ of 30 s, 60 ℃ of 1 min, 72 ℃ of 1 min, 30 circulations; 72 ℃ of 10 min, 4 ℃ of preservations.
Pcr amplification product detects with the agarose gel electrophoresis of 1.2 %, and except that used dna molecular amount standard was 100 bp DNA ladder Marker of the biochemical ltd of sky, Beijing root, all the other were with method 5.The result sees Fig. 2, and as can be seen from the figure, the Auele Specific Primer of c itrus canker germ go out the purpose band to ability PCR, and specific amplification is strong, explains that this method can obtain the DNA of high yield.
Claims (6)
1. the DNA water-bath of a c itrus canker germ precipitates process for extracting, it is characterized in that may further comprise the steps:
(a) new fresh thalli is added the lavation buffer solution thorough mixing, make the bacterial cell deposition, abandon supernatant;
(b) add lysis buffer in the deposition that makes toward step (a), vibration suspends, and in 65 ℃ of water-baths, complete reaction;
(c) extraction buffer of 65 ℃ of preheatings of adding, thorough mixing adds the saturated phenol of equal-volume, chloroform, the extracting of primary isoamyl alcohol mixing solutions, and the centrifugal albumen precipitation that makes sex change is got supernatant;
(d) be to add absolute ethyl alcohol in the supernatant that makes toward step (c) of the ratio of 1:2 according to volume ratio; After fully reaction is treated in 20 ℃ of placements of ﹣; High speed centrifugation to deposition is removed supernatant fully again, and adding volumetric concentration is the washing with alcohol deposition of 70 %, dries the back and adds sterilization ddH
2The O dissolution precipitation, 20 ℃ of preservations of ﹣ are subsequent use.
2. the DNA water-bath deposition process for extracting of c itrus canker germ according to claim 1 is characterized in that in the said step (a), adds lavation buffer solution 1 mL in per 0.12 g sample; Said lavation buffer solution consists of: 50 mmol/L Tris, and pH 7.7; 25 mmol/L EDTA; 0.1 % PVP.
3. c itrus canker germ DNA extraction method according to claim 1 is characterized in that in the said step (b), in the water-bath process, and water-bath time 5-10 min.
4. c itrus canker germ DNA extraction method according to claim 1 is characterized in that in the said step (b), adds lysis buffer 50 μ L in per 0.12 g sample; Said lysis buffer consists of: 50 mmol/L Tris, and pH 8.0; 25 mmol/L EDTA; 3.0 % SDS; 1.2 % PVP.
5. c itrus canker germ DNA extraction method according to claim 1 is characterized in that in the said step (c), adds extraction buffer 400 μ L in per 0.12 g sample; Said extraction buffer consists of: 50 mmol/L Tris, and pH 8.0; 25 mmol/L EDTA; 3.0 % SDS; 1.2 % PVP.
6. c itrus canker germ DNA extraction method according to claim 1, it is characterized in that in the said step (c) saturated phenol: chloroform: the primary isoamyl alcohol volume ratio is 25:24:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115044687A (en) * | 2022-06-17 | 2022-09-13 | 中国热带农业科学院三亚研究院 | Nested PCR (polymerase chain reaction) detection method for watermelon fruit blotch germs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115044687A (en) * | 2022-06-17 | 2022-09-13 | 中国热带农业科学院三亚研究院 | Nested PCR (polymerase chain reaction) detection method for watermelon fruit blotch germs |
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Application publication date: 20121003 |