CN101210032A - Simple and fast extraction method for DNA in grape wine - Google Patents
Simple and fast extraction method for DNA in grape wine Download PDFInfo
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- CN101210032A CN101210032A CNA2006101284665A CN200610128466A CN101210032A CN 101210032 A CN101210032 A CN 101210032A CN A2006101284665 A CNA2006101284665 A CN A2006101284665A CN 200610128466 A CN200610128466 A CN 200610128466A CN 101210032 A CN101210032 A CN 101210032A
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- dna
- silica
- grape wine
- supernatant liquor
- pure water
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- 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
Abstract
The invention discloses a simple and rapid DNA extraction method in wine, which comprises the following steps: A. 5g Silica is added with 5ml aqueous suspension for Silica precipitation to be evenly mixed after being cleaned by pure water and is set at the temperature of 4 DEG C for standby; B. wine with best adsorption conditions is then produced; C. the DNA of the wine is adsorbed and recycled: (1) Silica suspension is added to the wine mixture; (2) conservation is done for 1-2 hours under the temperature of 4DEG C, with constant shaking and oscillation; (3) centrifugation is done for 5 min under the temperature of 4DEG C with the amount of 5000g; (4) supernatant is removed and the Silica deposit is washed for 1-2 times by using washing fluid containing 0.1M Tris-HCl, pH 8.0, 1 percent of CTAB and 1M NaCl; (5) the centrifugation is done for 5 min with the amount of 5000g to collect the Silica deposit; (6) the Silica deposit is added with the pure water with the equivalent amount as the deposit and then well shaked and done with DNA heat elution after 5-10 min thermal insulation at the temperature of 65 DEG C; (7) centrifugation (10000g, 5min) is done to collect the supernatant containing DNA; (8) the supernatant in step (7) is extracted by chloroform with the same volume and the centrifugation (10000g, 3min) is done to collect top phase containing DNA; (9) the DNA in the supernatant is recycled and concentrated by using isopropanol precipitation; (10) the recycled wine DNA is then dissolved in 50 Mu lota TE (10mM Tris-HCl, pH 8.0) buffering agent.
Description
Technical field
The invention belongs to the technology of extracting DNA in the grape wine, specifically is exactly to extract the DNA analysis technology of grape wine DNA to be used to differentiate that grape wine is true and false.
Background technology
As everyone knows, grape wine is the wine kind of the unobstructed property in a kind of world, has very high nutritive value and health-care effect.The founder of wine fermentation theory, French big science man Louis. pasteur (L. Pasteur) was once said: " grape wine is the most healthy beverage ".Medical research shows, contains about 600 kinds of compositions to the human body beneficial in the grape wine, and trans-resveratrol wherein (resveratrol) has the effect (Jang et al.1997) of preventing cancer, reducing cholesterol.At present, drink people vinous and get more and more, famous-brand and high-quality red (in vain) grape wine is especially favored by common people.In many developed countries, drinking grape wine has become a kind of life fashion.
For a long time, various quality ginsengs vinous are inferior uneven on the market, and price does not wait from 2 dollars to 200 dollars, and existence is blent the Root-bark of Littlefruit Grape wine that forms by a certain percentage with alcohol, asccharin, grape essence, pigment etc.Quality vinous is mainly distinguished by veteran teacher of the sampling wine's sense organ, and common dealer and human consumer are difficult to distinguish the difference of famous-brand and high-quality grape wine and counterfeit and shoddy goods.In recent years, some famous grape wine companies and relevant research institution attempt to protect its famous and precious varietal wine, to win human consumer's trust by DNA analysis technical evaluation kind vinous in the world.For example, Australian Handry company is used for DNA antifalsification label mark their well-aged wine.Yet,, depend on the quality and quantity of the DNA that from grape wine, extracts to a great extent by the technology of DNA analysis evaluation varietal wine.
By following document as can be known, (Jang M, Cai L, Udeani GO et al. (1997) Cancerchemopreventive activity of resveratrol, a natural product from grapes.Science 275:218-220), (Rogstad SH. (2003) Plant DNA extraction usingSi lica.Plan tMol.Biol.Rep.21:463a-463g), (Siret R, Gigaud O et al. (2002) Analys i s of grape Vitis vinifera L. DNA in must mixtures andexperimental mixed wines using microsatellite markers.J.Agric.FoodChem.50 (13), 3822-3827), (Garc í a-Beneytez E, Moreno-Arribas MV et al. (2002) Application of a DNA analysis method for the cultivaridentification of grape musts and experimental and commercial wines ofVitisvinifera L.using microsatellite markers.J.Agric.Food Chem.50 (21), 6090-6096) with (SiretR, BoursiquotJM et al. (2000) Toward theauthentication of varietal wines by the analysis of grape (Vitis viniferaL.) residual DNA in must and wine using microsatellite markers.J.Agric.Food Chem.48 (10), 5035-5040), from red (in vain) grape wine, extract the method for DNA at present, all be by isopropanol precipitating (Siret R et al.2000,2002; Garc í a-Beneytez et al.2002).From new grape wine (must) or not relatively easy through extracting DNA the grape wine of clarifying treatment, because wherein may comprise broken grape tissue, can the normal DNA of extract phase from the precipitation of centrifugal collection.For the bottled grape wine of retailing because by fermentation, working procedures such as clarification, the DNA that derives from grape tissue is totally destroyed, trace amount DNA fragment certainty residual in the bottled grape wine is very little.Therefore, extract the grape wine that DNA needs large volume (500-1000ml) from the bottled grape wine of retail, conventional isopropanol precipitating method is not only operated inconvenience, and the DNA productive rate is low, poor repeatability.Therefore, it is imperative to set up a kind of simple and rapid technology of extracting DNA from grape wine, to satisfy the growing needs that utilize DNA analysis technical evaluation grape wine quality.
The main difficult point of extracting DNA from grape wine comprises: dna content is very little in (1) grape wine; (2) have a large amount of Polyphenols (tannin), polysaccharide, organic acid etc. in the grape wine, they seriously disturb DNA extraction and downstream to utilize the molecule experiment (as PCR) of DNA; (3) Chang Gui isopropanol precipitating method is used for grape wine operation inconvenience substantially; (4) DNA extraction is removed pollutents such as polyphenol before by dialysis, or utilizes lyophilize and the concentrated grape wine of centrifugation technique etc. time-consuming, and needs extra plant and instrument; (5) grape wine is slightly acidic, and generally between 3.4-4.2, this has also limited the application of conventional DNA extraction method to its pH.
Summary of the invention
In order to overcome the deficiencies in the prior art, the objective of the invention is to set up a kind of easy, quick, reliable method of from grape wine, extracting DNA, and the varietal wine that purification DNA is used for the PCR-based technology identified, so that the commercial famous and precious false proof dna molecular label that provides vinous is provided.
The present invention is achieved in that the simple and fast extraction method of DNA in the grape wine, may further comprise the steps:
A cleans up with the Silica of pure water with 5g, adds 5ml aqueous suspension Silica precipitation, mixing, put 4 ℃ standby;
B sets up the grape wine of optimal adsorption condition
Series preparation is regulated grape wine under slowly adding on the magnetic stirring apparatus:
1) to regulate pH vinous be about 8.0 to the NaOH solution that adds 10N;
2) add CTAB to 1% (w/v);
3) add NaCl to 1.0M;
4) add mercaptoethanol to 0.2% (v/v);
Absorption and the recovery of C grape wine DNA
(1) in above-mentioned grape wine mixed solution, adds Silica suspension;
(2) 4 ℃ following child care 1-2 hour, constantly rock, vibrate;
(3) centrifugal, 5000g, 5min, 4 ℃;
(4) abandon supernatant liquor, with containing 0.1M Tris-HCl, pH 8.0,1%CTAB, the of short duration flushing of the washing fluid of 1M NaCl Silica precipitation 1-2 time;
(5) centrifugal, 5000g, 5min collects the Silica precipitation;
(6) to the pure water of above-mentioned Silica precipitation adding equivalent, shake up, in 65 ℃ of hot eluted dnas of insulation 5-10min;
It is (7) centrifugal that (10000g 5min) collects the supernatant liquor that contains DNA;
(8) supernatant liquor of equal-volume chloroform extraction steps (7), centrifugal 10000g, 3min collects and contains upward phase of DNA;
(9) with DNA in the recovery of isopropanol precipitating method, the concentrated supernatant;
(10) the grape wine DNA of Hui Shouing is dissolved in 50 μ l TE (10mM Tris-HCl, pH 8.0) damping fluid.
Describedly clean up with the Silica of pure water with 5g, be meant 5g exsiccant Silica and 5ml pure water thorough mixing, room temperature sedimentation 5-10 hour is inhaled and is removed supernatant liquor, adds 5ml pure water thorough mixing again, and room temperature sedimentation 5-10 hour is inhaled and removed supernatant liquor.
The described Silica suspension that in above-mentioned grape wine mixed solution, adds; The amount by 5ml Silica/1000ml that is meant adds Silica suspension.
The supernatant liquor of described equal-volume chloroform extraction steps (7) is meant repetition chloroform extraction steps.
The supernatant liquor of described chloroform extraction steps (7), being meant increases the phenol extracting.
The present invention can use following method simultaneously;
Describedly clean up with the Silica of pure water with 5g, be meant 5g exsiccant Silica and 5ml pure water thorough mixing, room temperature sedimentation 5-10 hour is inhaled and is removed supernatant liquor, adds 5ml pure water thorough mixing again, and room temperature sedimentation 5-10 hour is inhaled and removed supernatant liquor;
The described Silica suspension that in above-mentioned grape wine mixed solution, adds; The amount by 5mlSilica/1000ml that is meant adds Silica suspension;
The supernatant liquor of described equal-volume chloroform extraction steps (7) is meant repetition chloroform extraction steps;
The supernatant liquor of described chloroform extraction steps (7), being meant increases the phenol extracting.
Principle of the present invention is to utilize the optionally characteristic of reversible adsorption dna fragmentation of Silica (silica, silicon dioxide).Interfering compounds such as the protein of non-specific adsorption to the Silica particle, polysaccharide, polyphenol can be removed by flushing, and the DNA of Silica absorption need just can desorption when heating.Under the upright top condition that the present invention determines, Silica can combine with the DNA of trace in red (in vain) grape wine efficiently, then by the Silica particle of centrifugal collection, thereby avoided the isopropanol precipitating method to operate the inconvenience of big quantity of fluid and poor repeatability, drawback that the DNA productive rate is low in conjunction with DNA.Although there are some to utilize Silica to extract the example (as Rogstad 2003) of DNA from plant leaf, the DNA of trace is different with DNA in the plant leaf in the grape wine, does not still have the report that utilizes Silica to extract DNA from grape wine both at home and abroad at present.
Easy, quick, the easy operation of method that the present invention sets up, and without any need for test kit; The DNA purity height (A260/280 is 1.8-2.0) that obtains, and productive rate height, repeatability is high.The Silica that is adopted, cheap, have no side effect.This invention is towards domestic and international winery and the institution of higher learning and the institute that are engaged in the research of grape, grape wine, is applicable to that the grape wine quality that relies on DNA analysis identifies and famous and precious dna molecular label technique vinous to have important use and be worth and economic benefit.
Embodiment
The pre-treatment Silica of 1Silica buys from (the product code name S5631 of Sigma-Aldrich company; On December 2nd, 2006, the price of inquiry was 509RMB/100g).5g exsiccant Silica and 5ml pure water thorough mixing, room temperature sedimentation 5-10 hour is inhaled and is removed supernatant liquor.Repeat above-mentioned washing process 1 time, add 5m l aqueous suspension Silica precipitation at last, mixing, put 4 ℃ standby.
The foundation of 2 optimal adsorption conditions
(1) to utilize the NaOH solution of 10N to regulate pH vinous be about 8.0 in the adjusting of grape wine pH, and this moment, its color just began to become blue.Colour-change is owing to the anthocyanin that derives from the grape pericarp (anthocynin);
(2) add CTAB to 1% (w/v) (10g/1000ml final volume);
(3) NaCl to 1.0M (58.44g/1000ml final volume);
(4) add mercaptoethanol to 0.2% (v/v) (2ml/1000ml final volume).
More than operation should slowly be carried out on magnetic stirring apparatus.
The absorption of 3 grape wine DNA and recovery
(1) in above-mentioned grape wine mixed solution, adds Silica suspension (5ml Silica/1000ml);
(2) 4 ℃ following child care 1-2 hour, constantly rock, vibrate;
(3) centrifugal, 5000g, 5min, 4 ℃ (the most handy centrifuge tube 500ml carries out on preparation type whizzer);
(4) abandon supernatant liquor, of short duration flushing Silica precipitates 1-2 time with washing fluid (contain 0.1M Tri s-HCl, pH 8.0,1%CTAB, 1M NaCl), to remove non-DNA class material (this step can be used the centrifuge tube of 30-50ml);
(5) centrifugal, 5000g, 5min collects the Silica precipitation;
(6) to the pure water of above-mentioned Silica precipitation adding equivalent, shake up, in 65 ℃ of hot eluted dnas of insulation 5-10min;
It is (7) centrifugal that (10000g 5min) collects the supernatant liquor that contains DNA;
(8) supernatant liquor of equal-volume chloroform extraction steps (7) is further removed pollutent; Centrifugal 10000g, 3min, phase in the collection (containing DNA) (can repeat the chloroform extraction steps in case of necessity, and increase the phenol extracting);
(9) DNA adopts conventional isopropanol precipitating method to reclaim, concentrate in the supernatant liquor;
(10) the grape wine DNA of Hui Shouing is dissolved in 50 μ l TE (10mM Tris-HCl, pH 8.0) damping fluid, and carries out quantitative, qualitative detection.
4DNA detects:
The ultraviolet spectrophotometry of can utilizing the grape wine DNA that purifies detects quantitatively, perhaps by agarose gel electrophoresis, ethidium bromide staining qualitative analysis (dyeing of susceptibility can be adopted SYBR Green1, Gel Red andGelGreen etc.).Molecular Detection is mainly utilized at the primer of the conservative region design of chloroplast(id) a/b binding-protein gene or is utilized microsatellite marker primer (as VVMD5), identifies by the special segment that pcr amplification produces whether the DNA that extracts derives from specific grape variety.
Claims (6)
1. the simple and fast extraction method of DNA in the grape wine may further comprise the steps:
A cleans up with the Silica of pure water with 5g, adds 5ml aqueous suspension Silica precipitation, mixing, put 4 ℃ standby;
B sets up the grape wine of optimal adsorption condition
Series preparation is regulated grape wine under slowly adding on the magnetic stirring apparatus:
1) to regulate pH vinous be about 8.0 to the NaOH solution that adds 10N;
2) add CTAB to 1% (w/v);
3) add NaCl to 1.0M;
4) add mercaptoethanol to 0.2% (v/v);
Absorption and the recovery of C grape wine DNA
(1) in above-mentioned grape wine mixed solution, adds Silica suspension;
(2) 4 ℃ following child care 1-2 hour, constantly rock, vibrate;
(3) centrifugal, 5000g, 5min, 4 ℃;
(4) abandon supernatant liquor, with containing 0.1M Tris-HCl, pH 8.0,1%CTAB, the of short duration flushing of the washing fluid of 1M NaCl Silica precipitation 1-2 time;
(5) centrifugal, 5000g, 5min collects the Silica precipitation;
(6) to the pure water of above-mentioned Silica precipitation adding equivalent, shake up, in 65 ℃ of hot eluted dnas of insulation 5-10min;
It is (7) centrifugal that (10000g 5min) collects the supernatant liquor that contains DNA;
(8) supernatant liquor of equal-volume chloroform extraction steps (7), centrifugal 10000g, 3min collects and contains upward phase of DNA;
(9) with DNA in the recovery of isopropanol precipitating method, the concentrated supernatant;
(10) the grape wine DNA of Hui Shouing is dissolved in 50 μ l TE (10mM Tris-HCl, pH 8.0) damping fluid.
2. the simple and fast extraction method of DNA in the grape wine as claimed in claim 1, it is characterized in that: describedly clean up with the Silica of pure water with 5g, be meant 5g exsiccant Silica and 5ml pure water thorough mixing, room temperature sedimentation 5-10 hour, supernatant liquor is removed in suction, add 5ml pure water thorough mixing again, room temperature sedimentation 5-10 hour is inhaled and is removed supernatant liquor.
3. the simple and fast extraction method of DNA in the grape wine as claimed in claim 1 is characterized in that: the described Silica suspension that adds in above-mentioned grape wine mixed solution; The amount by 5ml Silica/1000ml that is meant adds Silica suspension.
4. the simple and fast extraction method of DNA in the grape wine as claimed in claim 1 is characterized in that: the supernatant liquor of described equal-volume chloroform extraction steps (7) is meant repetition chloroform extraction steps.
5. the simple and fast extraction method of DNA in the grape wine as claimed in claim 1 is characterized in that: the supernatant liquor of described chloroform extraction steps (7), being meant increases the phenol extracting.
6. the simple and fast extraction method of DNA in the grape wine as claimed in claim 1 is characterized in that: the present invention can use following method simultaneously:
A is described to be cleaned up with the Silica of pure water with 5g, is meant 5g exsiccant Silica and 5ml pure water thorough mixing, and room temperature sedimentation 5-10 hour is inhaled and removed supernatant liquor, adds 5ml pure water thorough mixing again, and room temperature sedimentation 5-10 hour is inhaled and removed supernatant liquor;
The described Silica suspension that in above-mentioned grape wine mixed solution, adds of b; The amount by 5mlSilica/1000ml that is meant adds Silica suspension;
The supernatant liquor of the described equal-volume chloroform of c extraction steps (7) is meant repetition chloroform extraction steps;
The supernatant liquor of the described chloroform extraction steps of d (7), being meant increases the phenol extracting.
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| CN2006101284665A CN101210032B (en) | 2006-12-26 | 2006-12-26 | Extraction method for DNA in grape wine |
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| CN2006101284665A CN101210032B (en) | 2006-12-26 | 2006-12-26 | Extraction method for DNA in grape wine |
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| CN101210032B CN101210032B (en) | 2011-08-31 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011067630A1 (en) | 2009-12-04 | 2011-06-09 | Universidade De Trás-Os-Montes E Alto Douro | Method and kit for dna extraction from vitis vinifera l. and for amplification and detection of grapevine varieties or cultivars in musts or wines |
| CN104450683A (en) * | 2014-12-22 | 2015-03-25 | 广东出入境检验检疫局检验检疫技术中心 | Method for extracting grape genome DNA from wine |
| WO2020109597A1 (en) | 2018-11-30 | 2020-06-04 | Orvinum Ag | Method for providing an identifier for a product |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19856064C2 (en) * | 1998-12-04 | 2000-11-30 | Invitek Gmbh | Universal method for the isolation of DNA from any starting material |
| CN100441686C (en) * | 2005-12-30 | 2008-12-10 | 华中农业大学 | Small quality fast extraction method for soil total DNA |
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2006
- 2006-12-26 CN CN2006101284665A patent/CN101210032B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011067630A1 (en) | 2009-12-04 | 2011-06-09 | Universidade De Trás-Os-Montes E Alto Douro | Method and kit for dna extraction from vitis vinifera l. and for amplification and detection of grapevine varieties or cultivars in musts or wines |
| CN104450683A (en) * | 2014-12-22 | 2015-03-25 | 广东出入境检验检疫局检验检疫技术中心 | Method for extracting grape genome DNA from wine |
| CN104450683B (en) * | 2014-12-22 | 2017-07-14 | 广东出入境检验检疫局检验检疫技术中心 | A kind of method that grape genomic DNA is extracted from grape wine |
| WO2020109597A1 (en) | 2018-11-30 | 2020-06-04 | Orvinum Ag | Method for providing an identifier for a product |
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| CN101210032B (en) | 2011-08-31 |
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