CN203007255U - Quick extracting device for pine wood nematode nucleic acid in wood - Google Patents
Quick extracting device for pine wood nematode nucleic acid in wood Download PDFInfo
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- CN203007255U CN203007255U CN 201220581808 CN201220581808U CN203007255U CN 203007255 U CN203007255 U CN 203007255U CN 201220581808 CN201220581808 CN 201220581808 CN 201220581808 U CN201220581808 U CN 201220581808U CN 203007255 U CN203007255 U CN 203007255U
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- nucleic acid
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- wood nematode
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Abstract
The utility model discloses a quick extracting device for pine wood nematode nucleic acid in wood. The quick extracting device comprises a wood collecting device and a nucleic acid quick extracting device which are used in match; a wood obtaining device comprises a collector, a drill bit and an electric drill; the nucleic acid quick extracting device comprises an injector, a filter, an adsorption column and an absorption head; an adsorption film is placed in the adsorption column; the drill bit is connected with the electric drill while collecting the bits of wood, and the collector is arranged between the drill bit and the wood to be measured, wherein the injector is arranged below the collector; and when in extracting and computing, the injector is connected with the absorption head to absorb the lysis solution, and then is sequentially connected with the filter, the adsorption column and the absorption head to extract the nucleic acid. By adopting the quick extracting device for the pine wood nematode nucleic acid in the wood, the nucleic acid in a sample can be directly and quickly extracted, the instrument and equipment such as a centrifugal machine can be saved during extracting, plenty of time is saved, and a plurality of operation steps are simplified; and the quick extracting device is low in cost, quick to operate, and high in practicability.
Description
Technical field
The utility model belongs to technical field of molecular biology, is specifically related to pine wood nematode nucleic acid rapid extraction device in a kind of timber.
Background technology
Detecting at present the pine wood nematode method mainly contains following several:
(1) morphology differential method: the doubtful wood cutting tool that pine wood nematode is arranged that dyes is rived into little strip, then strip timber is placed in lower hopper shape Glass Containers, rubber tubing of lower hopper exit cover at glassware, adding a spring pinchcock on pipe clamps, adding water in Glass Containers soaks completely batten, static a couple of days, make pine wood nematode dissociate from timber in water and sink to funnel exit, decontrol spring pinchcock the water that the bottom contains worm is put into glass dish, examine under a microscope, with morphological feature diagnosis pine wood nematode.The disadvantage of this method is, length consuming time, efficient are low, morphology differentiates to have uncertainty.In Japan, Mamiya etc. just proposed to adopt the tail point of afterbody to dash forward to distinguish pine wood nematode and intend pine wood nematode as far back as 1979, and this is also the Main Morphology foundation during present two kinds of nematodes are differentiated.Because the female worm afterbody in pine wood nematode some colony of North America also has the tail point prominent, it is very difficult therefore both right areas will being separated some amateur personnel that are engaged in the Plant nematode classification on the form.
(2) enzyme assay: glutamic oxaloacetic transaminase (GOT) is the enzyme that can distinguish two kinds of nematodes, the bands of a spectrum type of the cellulase of pine wood nematode secretion is different from the cellulase of intending pine wood nematode, Esterase Isozyme and malate dehydrogenase zymogram also can be distinguished pine wood nematode and intend pine wood nematode.Because the stability of esterase is also bad, same strain is also changing after cultivating on different bacterium.This may with enzyme itself be subject to external environmental condition as holding conditions, analytical procedure and the nematode of condition (humidity and temperature etc.), leaching process and the extract of cultivating nematode itself be nematode each etap or a certain stage nematode the impact of physiological situation relevant.Therefore, although enzyme is keeping high conservative between closely-related kind, due to above-mentioned factor, utilize electrophoretic analysis nematode albumen easily to cause the unstable of result.The method is complicated, loaded down with trivial details, unstable, poor specificity, without actual application value.
(3) immunological method: mainly contain polyclonal antibody and monoclonal antibody technique, measure with polyclonal antibody enzyme-linked immunization, although the susceptibility of EL ISA is high, the species separation degree is also bad, and this poor specificity with polyclonal antibody is relevant.With monoclonal anti body method existing successful precedent in the evaluation of some important plant nematodes, tell globodera rostochiensis (Globodera rostochiensis) and G.pallida (G.pallida) as the using monoclonal antibody such as Arjen Schots technical area, other also had the application of monoclonal antibody technique as soy bean cyst roundworm (Het2erodera glycines) and Meloidogyne incognita (M. incog2nita).
(4) hormone determination: pine wood nematode with intend the sex pheromone that pine wood nematode produces and have species specificity, i.e. pine wood nematode or intend pine wood nematode and only have female worm separately could attract separately male worm accordingly, this is the reaction that causes of female worm release property pheromone separately.Therefore can utilize mensuration sex pheromone to distinguish both.Pine wood nematode with intend the sex pheromone that pine wood nematode produces and have species specificity, i.e. pine wood nematode or intend pine wood nematode and only have female worm separately could attract separately male worm accordingly, this is the reaction that causes of female worm release property pheromone separately.Therefore can utilize mensuration sex pheromone to distinguish both.
(5) lipid assay method: Krusberg etc. (1973) report, the ripe female worm of Meloidogyne incognita and peanut root-knot nematode is identical with the lipid acid character of ovum, but in two kinds of nematodes, the content of certain lipid acid is different, therefore can distinguish two kinds of nematodes with this.But have not yet to see useful lipid and distinguish pine wood nematode and the report of intending pine wood nematode, whether feasible in this respect, also can carry out attempting.
(6) nucleic acid determination method: along with the fast development of Protocols in Molecular Biology in recent years, take the diagnosis of DNA as the basis, the effect in the nematode classification is identified grows with each passing day, and has set up more ripe molecular diagnosis system in many Plant nematode monoids.The test kit supply of PCR and the RT-qPCR of comparative maturity has been arranged at present.
In the nucleic acid determination method, the method for extracting nucleic acid from biology is a lot of, and the method for extracting at present nucleic acid mainly contains following several:
(1) organic extraction method.Ultimate principle: phenol makes protein denaturation, SDS lysing cell film, and Proteinase K and EDTA digestible protein or polypeptide or little peptide, nucleoprotein sex change degraded dissociates out DNA from nucleoprotein.Utilize DNA soluble in water, be insoluble to the feature extraction DNA of organic solvent.
(2) resin method.Ultimate principle: as example, it contains paired Iminodiacetate ion, and high volence metal ion is had very high avidity and sequestering action with the main resin chelex-100 that uses.Can make the cytolemma cracking in low ionic strength, alkalescence and under boiling, and make protein denaturation, DNA is free.
(3) inorganic extraction method.Ultimate principle: utilize NaCI or sodium-acetate protein precipitation, through centrifugal, extract DNA with the dehydrated alcohol precipitation.
(4) difference cracking extraction method.Ultimate principle: utilize the cytolemma of some biological specimen to be easy to fragmentation, the cell of other biological specimens is not allowed breakable characteristics, uses ordinary method first to remove and is easy to smudge cells, then be not easy smudge cells with the special reagent cracking, then extract DNA wherein.
(5) phosphate buffered saline buffer method.Ultimate principle: after cell digested in the solution that contains nonionic washing agent and Proteinase K, heat made protein and Proteinase K inactivation, and the centrifugation isolated protein extracts DNA from centrifuged supernatant.
(6) methane amide depolymerization.Ultimate principle: the mixture of methane amide cleavable DNA and protein, mutability precipitates the protein that discharges again, but the activity of Proteinase K is not made significant difference.With DNA and protein in highly difficult methane amide cracking chromatin, then carry out fully dialysis with the guncotton poly-bag and remove Proteinase K and organic solvent.The pulsed-field gel electrophoresis analysis that this method is suitable for building the genome dna library of heavy body carrier and carries out large fragment.
(7) glass stick winding method.Ultimate principle: DNA can be deposited in the interface of cell pyrolysis liquid and ethanol, large fragment DNA is transferred to from dehydrated alcohol in the TE liquid of PH8.0 with the glass stick of buckle.This method is applicable to build gene library, and pcr amplification is suitable for extracting DNA simultaneously from different cell or tissue samples.
(8) based on the method for silicon-dioxide adsorbing and extracting nucleic acid.Ultimate principle: the characteristic of utilizing silicon-dioxide absorption nucleic acid, organism is through guanidinium isothiocyanate, nucleic acid and silica bound after the Tris-HCl-EDTA of roton X-100 processes, Tris-HCl damping fluid through containing guanidinium isothiocyanate and 70% ethanol and acetone wash respectively again, use at last the TE wash-out, obtain nucleic acid.
(9) glass fibre membrane absorption method.Ultimate principle: glass fibre membrane has the advantages that to be combined with the nucleic acid reversibility, DNA in the organism lysate is combined with glass fibre membrane under the effect of short adsorption liquid, remove albumen and other impurity that adsorbs on glass fibre membrane with protein liquid removal, again clean with washings albumen and other impurity that adsorbs on glass fibre membrane again, with elutriant, the nucleic acid that adsorbs on glass fibre membrane is eluted at last.
(10) paramagnetic particle method.Ultimate principle: utilize the strong protein denaturants such as guanidine thiocyanate, destroy cytolemma and nuclear membrane albumen, released dna, and make the nuclease inactivation; Then add magnetic bead to pass through chemical group and the DNA specific adsorption on surface, and the impurity such as protein are not adsorbed and stay and hold night; Then under the effect in magnetic field, magnetic-particle separates with liquid, reclaims particle; Last again with the DNA of pure water or the absorption of TE wash-out, carry out dissociating of DNA and magnetic bead in lysate, with DNA stripping again.
(11) pellosil adsorption column extraction method.Ultimate principle: silica gel mould adsorption column method is the DNA that adopts special silica gel mould adsorption column to come the selective adsorption lysing cell to discharge, then through simple programs such as washing and wash-outs, just can obtain high purity DNA.
At present, aspect nucleic acid extraction, eliminate gradually certain operations complexity and loaded down with trivial details method in the world.That glass fibre membrane absorption method and pellosil adsorption column extraction method have is easy and simple to handle, easily obtain high purity DNA with RNA, can adapt to by the thickness of regulating adsorption film that the nucleic acid that extracts different amounts requires and the advantage such as with low cost, so be widely used in various nucleic acid determination test kits.
The utility model content
Goal of the invention: for the deficiencies in the prior art, the purpose of this utility model is to provide pine wood nematode nucleic acid rapid extraction device in a kind of timber so that its have efficiently, the advantage such as rapid extraction pine wood nematode nucleic acid.
Technical scheme: for achieving the above object, the technical solution adopted in the utility model is as follows:
Pine wood nematode nucleic acid rapid extraction device in a kind of timber comprises that the timber collection device and the nucleic acid speed that are used in conjunction with each other carries device; Described timber deriving means comprises collector, drill bit and electric drill; Described nucleic acid speed is carried device and is comprised syringe, strainer, adsorption column and suction nozzle, establishes adsorption film in adsorption column; When collecting wood chip, described drill bit is connected with electric drill, and described collector is located between drill bit and timber to be measured; Establish syringe below described collector; When extracting accounting, described syringe first is connected with suction nozzle and draws lysate, then is connected with strainer, adsorption column and suction nozzle and is connected extraction nucleic acid.
Described collector is the T-shaped three-way collector of left and right both ends open, lower ending opening.
Described drill bit adopts the timber opening bit, and its anterior diameter is 10 ~ 40mm.
Described electric drill is manual infinite variable speed hand held electric drill, adopts lithium battery power supply.
Described adsorption film is selected from glass fibre membrane, pellosil; Thickness 0.1 ~ the 10mm of adsorption film.
The capacity of described syringe is in 1 ~ 100mL scope.
Described strainer is the hollow circular container that contains upper and lower joint made of plastic, and top connection is connected with syringe nozzle, and lower sub is connected with adsorption column, places filtering membrane in bulge.
The internal diameter of described strainer is 5 ~ 50mm.
In described strainer, filtering membrane is selected from blend fiber film, cellulose acetate film, nitrocellulose membrane, nylon membrane, poly (ether sulfone) film or glass fibre membrane; The thickness of filtering membrane is 0.1 ~ 10mm, and the aperture of filtering membrane is 0.1 ~ 10um.
All scribble silicone oil on described adsorption column inwall, suction nozzle inwall.
Pine wood nematode nucleic acid rapid extraction device in timber of the present utility model, use electric drill and increment borer head, directly timber is collected in syringe by the timber collection device, then directly draws the pine wood nematode lysate with syringe and heat, make the pine wood nematode cracking discharge nucleic acid.The lysate that contains nucleic acid can be directly by being connected in the filter membrane in the strainer on syringe, particle and the impurity in the filtering sample effectively, and nucleic acid is not adsorbed by filter membrane.Directly by being connected in simultaneously the nucleic acid adsorption column on strainer, the nucleic acid in liquid is adsorbed the adsorption film absorption in post, by cleaning, the nucleic acid wash-out is increased and detect with elutriant at last by the continuation of the liquid after strainer.
Beneficial effect: compared with prior art, pine wood nematode nucleic acid rapid extraction device in timber of the present utility model, can directly extract rapidly the nucleic acid in sample, leaching process need not to use the plant and instrument such as whizzer, has saved the plenty of time, has simplified many operation stepss, cost is low, operation is fast, has good practicality, can produce good economic benefit and social effect.
Description of drawings
Fig. 1 is the structural representation of timber collection device;
Fig. 2 is the structural representation that nucleic acid speed is carried device.
Embodiment
Further illustrate the utility model below in conjunction with accompanying drawing, this embodiment is implemented under take technical solutions of the utility model as prerequisite, should understand these modes and only be used for explanation the utility model and be not used in restriction scope of the present utility model.
As depicted in figs. 1 and 2, pine wood nematode nucleic acid rapid extraction device in timber comprises that timber collection device and nucleic acid speed carries device.The timber deriving means comprises collector 11, drill bit 12 and electric drill 13.Nucleic acid speed is carried device and is comprised syringe 21, strainer 22, adsorption column 23 and the suction nozzle 25 that is connected successively, establishes adsorption film 24 in adsorption column 23.Collector 11 is the T-shaped three-way collector of left and right both ends open, lower ending opening; Drill bit 12 adopts the timber opening bit, and its anterior diameter is 10 ~ 40mm; Electric drill 13 is manual infinite variable speed hand held electric drill, adopts lithium battery power supply; Syringe 21 is connected with collector 11 lower ending openings.The capacity of syringe 21 is in 1 ~ 100mL scope.Strainer 22 is the hollow circular container that contains upper and lower joint made of plastic, top connection is connected with 21 of syringes, lower sub is connected with adsorption column 23, place filtering membrane in bulge, filtering membrane is selected from blend fiber film, cellulose acetate film, nitrocellulose membrane, nylon membrane, poly (ether sulfone) film or glass fibre membrane; The thickness of filtering membrane is 0.1 ~ 10mm, and the aperture of filtering membrane is 0.1 ~ 10um; The internal diameter of strainer 22 is 5 ~ 50mm.Adsorption film 24 is selected from glass fibre membrane, pellosil; Thickness 0.1 ~ the 10mm of adsorption film.All scribble silicone oil on adsorption column 23 inwalls, suction nozzle 25 inwalls.
During use, at first determine tested timber 15, be close to timber 15 surfaces with the opening end on T-shaped applicator 11 tops, collector 11 lower ends connect syringe 21, then pass collector 11 tops with the electric drill 13 that drill bit 12 is housed, finger is pressed the electric drill switch and is drilled through wood chip, and wood chip can flow in syringe 21 automatically.Finger can arbitrarily be regulated the speed of electric drill by the control elasticity of electric drill switch, controls the pressure between drill bit and timber, can obtain the different wood chip of thickness.
Syringe 21 heads that will contain the wood chip sample are loaded onto suction nozzle 25, draw the lysate (TES+ Proteinase K) of different amounts according to the amount of wood chip, and 65 ℃ of cracking 30min heat.Then suction nozzle 25 is removed, strainer 22 is contained in syringe 21 heads, then nucleic acid adsorption column 23 is connected on strainer 22.Syringe 21, strainer 22 and adsorption column 23 are connected.Then use hand propelled syringe shank, the lysate in wood chip is pushed strainer 22, and enter adsorption column 23 through strainer 22, at last the lysate release is discarded.Through this process, the cleaved DNA that discharges of pine wood nematode in wood chip, impurity in lysate is cut by strainer 22, and the DNA in lysate can adsorb by the adsorption film 24 that strainer is adsorbed in post 23, again syringe 21 and strainer 22 are sloughed, clean adsorption film 24 with scavenging solution (75% ethanol) and further go out decon, (1 * TE) collects the DNA wash-out in adsorption film 24 in the centrifuge tube of lid, for amplification to use at last the nucleic acid elutriant.
Claims (10)
1. pine wood nematode nucleic acid rapid extraction device in a timber is characterized in that: comprise that the timber collection device and the nucleic acid speed that are used in conjunction with each other carries device; Described timber deriving means comprises collector (11), drill bit (12) and electric drill (13); Described nucleic acid speed is carried device and is comprised syringe (21), strainer (22), adsorption column (23) and suction nozzle (25), establishes adsorption film (24) in adsorption column (23); When collecting wood chip, described drill bit (12) is connected with electric drill (13), and described collector (11) is located between drill bit (12) and timber to be measured (15); Establish syringe (21) below described collector (11); When extracting accounting, described syringe (21) first is connected with suction nozzle (25) and draws lysate, then is connected 25 with strainer (22), adsorption column (23) with suction nozzle) be connected successively and extract nucleic acid.
2. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1 is characterized in that: described collector (11) is the T-shaped three-way collector of left and right both ends open, lower ending opening.
3. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1 is characterized in that: described drill bit (12), adopt the timber opening bit, and its anterior diameter is 10 ~ 40mm.
4. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1, it is characterized in that: described electric drill (13) is manual infinite variable speed hand held electric drill, adopts lithium battery power supply.
5. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1, it is characterized in that: described adsorption film (24) is selected from glass fibre membrane, pellosil; Thickness 0.1 ~ the 10mm of adsorption film.
6. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1, it is characterized in that: the capacity of described syringe (21) is in 1 ~ 100mL scope.
7. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1, it is characterized in that: described strainer (22) is the hollow circular container that contains upper and lower joint made of plastic, top connection is connected with syringe (21) head, lower sub is connected with adsorption column (23), places filtering membrane in bulge.
8. pine wood nematode nucleic acid rapid extraction device according to claim 1 or 7 described timber, it is characterized in that: the internal diameter of described strainer (22) is 5 ~ 50mm.
9. pine wood nematode nucleic acid rapid extraction device according to claim 1 or 7 described timber is characterized in that: in described strainer (22), filtering membrane is selected from blend fiber film, cellulose acetate film, nitrocellulose membrane, nylon membrane, poly (ether sulfone) film or glass fibre membrane; The thickness of filtering membrane is 0.1 ~ 10mm, and the aperture of filtering membrane is 0.1 ~ 10um.
10. pine wood nematode nucleic acid rapid extraction device in timber according to claim 1, is characterized in that: all scribble silicone oil on described adsorption column (23) inwall, suction nozzle (25) inwall.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201220581808 CN203007255U (en) | 2012-11-07 | 2012-11-07 | Quick extracting device for pine wood nematode nucleic acid in wood |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201220581808 CN203007255U (en) | 2012-11-07 | 2012-11-07 | Quick extracting device for pine wood nematode nucleic acid in wood |
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| CN203007255U true CN203007255U (en) | 2013-06-19 |
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| CN 201220581808 Expired - Fee Related CN203007255U (en) | 2012-11-07 | 2012-11-07 | Quick extracting device for pine wood nematode nucleic acid in wood |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965271A (en) * | 2012-11-07 | 2013-03-13 | 苏州市外来有害生物防控技术中心 | Device for quickly extracting pine wood nematode nucleic acid |
| CN106434639A (en) * | 2016-12-01 | 2017-02-22 | 山东森芃生物科技有限公司 | DNA extracting method needing no tube transferring |
-
2012
- 2012-11-07 CN CN 201220581808 patent/CN203007255U/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965271A (en) * | 2012-11-07 | 2013-03-13 | 苏州市外来有害生物防控技术中心 | Device for quickly extracting pine wood nematode nucleic acid |
| CN102965271B (en) * | 2012-11-07 | 2014-05-28 | 苏州市外来有害生物防控技术中心 | Device for quickly extracting pine wood nematode nucleic acid |
| CN106434639A (en) * | 2016-12-01 | 2017-02-22 | 山东森芃生物科技有限公司 | DNA extracting method needing no tube transferring |
| CN106434639B (en) * | 2016-12-01 | 2018-07-20 | 山东森芃生物科技有限公司 | A kind of DNA extraction method without tube |
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Granted publication date: 20130619 Termination date: 20131107 |