CN101570799B - Chip for screening viruses of cucumovirus and application thereof - Google Patents
Chip for screening viruses of cucumovirus and application thereof Download PDFInfo
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- CN101570799B CN101570799B CN2009100865462A CN200910086546A CN101570799B CN 101570799 B CN101570799 B CN 101570799B CN 2009100865462 A CN2009100865462 A CN 2009100865462A CN 200910086546 A CN200910086546 A CN 200910086546A CN 101570799 B CN101570799 B CN 101570799B
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Abstract
The invention discloses a chip for screening viruses of cucumovirus and application thereof. The chip is a DNA chip which fixes at least 16 DNA fragment arrays on a carrier surface, wherein the nucleotide sequences of the 16 DNA fragment arrays are sequences 1 to 16 in the sequence table, respectively. The method analyzes the nucleotide sequences of the cucumovirus by bioinformatics method, designs compatible probes for the cucumovirus, and by standard virus source verification, establishes a set of chip-based screening technical system of emerging viruses of the cucumovirus in the world, and effectively overcomes the defects of the existing monitoring methods, thus strengthening the ability of early warning the emerging viruses of the cucumovirus.
Description
Technical field
The present invention relates to the chip and the application thereof of screening viruses of cucumovirus.
Background technology
The present invention relates to information biology and biological detection authenticate technology field, relate in particular to Cucumovirus is known and unknown virus is identified probe and operating system.Be mainly used in the Cucumovirus virus quarantine of customs examination and quarantine system, the diagnosis of the plant protection unit viroses of plant and the fields such as virus evaluation in the plant virus means of taxonomic research.
According to (the International Committee on Taxonomy of Viruses of ICTV, ICTV) the 8th subseries report, Cucumovirus (Cucumovirus) has 3 kinds to determine virus, be cucumber mosaic virus (Cucumber mosaic virus, CMV), peanut stunt virus (Peanut stunt virus, PSV), tomato aspermy virus (Tomato aspermy virus, TAV).The representative species cucumber mosaic virus has very wide host range, can infect the dicotyledonous and monocotyledons of kind more than 1000, it is one of topmost pathogenic virus on China's Cruciferae, Solanaceae, pulse family and the cucurbitaceae vegetable, also be the important cause of disease of tobacco, banana, Herba Passiflorae Caeruleae, also have many flowers, medicinal plant and ruderal also to be subjected to this virus infection.At present, China is separated to this virus from 120 various plants of 39 sections.Peanut stunt virus is the quarantine venereal disease poison in 2007 issue " People's Republic of China (PRC) enter the territory Plant Quarantine harmful organism register ".
Because this accessory has the possibility of potential New Development quarantine venereal disease poison, exist multiple grouping simultaneously, the omission false retrieval appears in specific antiserum(antisera) and primer easily when detecting the evaluation known viruse, and can't identify the unknown virus that may exist in this genus, thereby the possibility that causes dangerous virus to propagate diffusion increases, and causes the risk of financial loss and social influence to increase.
The detection method that is used for Cucumovirus virus at present comprises biological method, immunological method, Electron Microscopy, Protocols in Molecular Biology etc., these methods are primarily aimed at the Cucumovirus known viruse and carry out specific detection, and are powerless for the monitoring of virus the unknown, New Development.
Summary of the invention
The purpose of this invention is to provide a kind of chip and application thereof of screening viruses of cucumovirus.
The chip of screening viruses of cucumovirus provided by the present invention is a DNA chip of fixing 16 kinds of dna fragmentation arrays at carrier surface, and the nucleotide sequence of described 16 kinds of dna fragmentation arrays is respectively sequence 1 in the preface in the sequence table to sequence 16.
Another object of the present invention provides a kind of method of screening viruses of cucumovirus.
The method of screening viruses of cucumovirus provided by the present invention is to carry out Cucumovirus virus in the detection of expression plant with described chip.
Probe in the chip of screening viruses of cucumovirus of the present invention has Cucumovirus and belongs to specificity in highly compatible on the level and the Tobamovirus, finish the examination for the treatment of the New Development virus that may exist in the sample product in a few hours, required sample size is few, generally only needs 0.1g.The analysis of data combines with Computer Image Processing software in addition, and reaching analytical results can visualize, visual.
The present invention adopts bioinformatics method that the nucleotide sequence of Cucumovirus is analyzed, and has designed the compatible probe of this genus, and standard poison source checking result proves that probe is respond well.The chip of screening viruses of cucumovirus of the present invention can be used for the diagnosis or the plant virus of the quarantine of Cucumovirus virus, the viroses of plant to be identified.
Description of drawings
Fig. 1 is a Cucumovirus virus examination chip probe dot matrix (7 * 9).
Fig. 2 is for using the result of TAV standard poison source checking Cucumovirus examination chip.
Fig. 3 is for using the result of CMV standard poison source checking Cucumovirus examination chip.
Fig. 4 is an examination chip sensitivity detected result.
Fig. 5 is a PCR sensitivity detected result.
Embodiment
The chip of embodiment 1, screening viruses of cucumovirus
One, the chip of preparation screening viruses of cucumovirus
1, belongs to the design of level highly compatible oligonucleotide probe
1) downloads the full genome of Cucumovirus and nucleic acid sequence data is used for designing probe from the U.S. state-run biotechnology information center (NCBI) and the international virusology classification council (ICTV) database.
2) use viral whole genome sequence and whole nucleotide sequence designing probe respectively.Need screen when using whole nucleotide sequence, remove nucleotide sequence less than 160bp length.If the whole nucleotide sequence numbers of this Tobamovirus greater than 300, then use the sequence alignment program BLASTN of NCBI that nucleotide sequence is gone redundancy, if promptly certain nucleotide sequence 90% above length and other sequences have 95% similarity, this sequence of deletion from database then.
3) download whole plant mRNA sequence datas, all animals virus genome sequence data and bacterial genomes sequence that can infection plant from NCBI RefSeq database, be used for specific data storehouse as probe design.
4) use the BLASTN connection to join sequence in the Tobamovirus, seek sequence conservative region designing probe.During probe design in the sequence conservative region with 5 bases as at interval, extract the nucleotide sequence of all 70bp continuously, the nucleotide sequence of choosing is screened, standard is that GC content is between 40% and 60%, any single base contents is not more than 50%,, do not reach the hairpin structure that can not form greater than 8bp greater than the continuous repetition base of 5bp.
5) using other all plant virus nucleic acid sequences of specific data storehouse that sequence that BLASTN will be by the step 4) screening and step 3) obtain and non-this Tobamovirus to carry out specificity compares, screening criteria is joined sequence length for connection and be multiply by similarity less than 25bp, and does not have the above sequences match that fits like a glove of 20bp.
6) use BLASTN to compare by the probe sequence of step 5) screening and all virus sequences of this Tobamovirus, the comparison standard is joined sequence length for connection and be multiply by similarity greater than 56bp, and the sequences match of continuous 15bp is arranged.Satisfy the sequence of this standard, be the probe that can detect corresponding virus.
7) use (the Institute for Theoretical Chemistry of University of Vienna theoretical chemistry institute, University of Vienna) the RNAcofold program of exploitation is calculated probe and corresponding virus sequence bonded free energy, and self forms the free energy of RNA secondary structure.
8) probe sequence that step 5) is obtained sorts, order standard is followed successively by probe to all viral fraction of coverage of this Tobamovirus, probe combines total free energy with this Tobamovirus sequence, probe combines total similarity with this Tobamovirus virus sequence, the non-specific combination similarity in probe and specific data storehouse, probe forms self RNA secondary structure free energy.
9) probe that step 5) is obtained goes redundancy, removes the repetition probe, makes that maximum repeat length is no more than 25bp between the probe.The multiple probe only keeps best probe according to the standard sorted of step 8).The probe that this step obtains is non-repetitive specific probe.
10) standard set by step 8) sorts to the probe that step 9) obtains, and selects probe successively according to ranking results, makes each virus sequence of this Tobamovirus have 5 probe correspondences at least.During selection,,, pay the utmost attention to the fraction of coverage that other is not reached the virus sequence of 5 probe correspondences, sort according to the standard of step 8) more afterwards then to the ordering of other non-selected probes if some virus sequence has reached 5 probe correspondences.If the process step 9) can't reach the standard of at least 5 probe correspondences of each virus sequence of this Tobamovirus, then reduce the sequence conservation standard in step 3), redesign, circulating according to this all has 5 probe correspondences up to all virus sequences.If can't be from the conservative region designing probe, or sequence have conservative region in the Tobamovirus, then uses all virus sequence full length sequence designing probes.The probe design standard is identical to step 9) with step 4).
11) probe validity coefficient is defined as number of probes divided by the nucleic acid sequence number, the validity coefficient is 5 to the maximum, and minimum is 5 divided by the sequence number.This coefficient is used to represent whether sequence is conservative in the Tobamovirus, if promptly coefficient is 5, then represents no conserved sequence in this Tobamovirus, and coefficient is more little, and conservative property is high more between the sequence.Select probe that the Tobamovirus genome sequence designs or whole probes of designing of nucleotide sequence according to the validity coefficient, select the validity coefficient littler, and number of probes is more near 50 the probe groups probe groups as this Tobamovirus.
Designed 16 probes according to mentioned above principle, the nucleotide sequence of 16 probes is shown in sequence 1-16 in the sequence table.
2, the preparation of chip
Above-mentioned 16 probes dissolve with the point sample damping fluid, and concentration is 50 μ M, and every probe laterally repeats 3 points, every about 0.25nL, and the about 180 μ m of spot diameter, dot spacing is 300 μ m, the standard variance of point sample uniformity coefficient is 15%.The chip point is had one side hydration 2～10s on 65 ℃ of water-baths of probe, chip is 2-3cm apart from water surface distance, in the air at room temperature seasoning, is carrying out a hydration.The one side that point is had a probe upwards, it is crosslinked to be placed in the UV-crosslinked instrument 250mJ.Chip is placed on 42 ℃ of preheatings, and 0.5%SDS cleans 10min.Chip transferred in 42 ℃ of pre-hot distilled waters clean 2min.Chip is placed in the 50ml taper centrifuge tube, and the centrifugal 1min of 2000rpm/min to remove the liquid of chip surface, obtains the chip of screening viruses of cucumovirus.
Wherein the chip of a screening viruses of cucumovirus as shown in Figure 1,1-16 is a Cucumovirus examination chip probe among Fig. 1, Hex is a fixedly positive quality control of chip, PC for hybridization positive quality control, NC for hybridization negative Quality Control, BC is blank Quality Control.
3, being used to the total RNA of the sample that detects extracts
1) get the 0.2g sample, powdered with liquid nitrogen grinding, move in the 1.5ml centrifuge tube of sterilization, add the Trizol reagent of 1ml then, concuss shakes up;
2) 4 ℃, the centrifugal 10min of 12000rpm to be removing insoluble composition, and supernatant liquor is changed in the new 1.5ml centrifuge tube;
3) keep 5min under the room temperature, add the 0.5ml chloroform, thermal agitation 15s, at room temperature keep 2-15min then after, 4 ℃, the centrifugal 15min of 12000rpm;
4) with the upper water phase transition in new 1.5ml centrifuge tube, add the equal-volume Virahol, put upside down mixing, keep 15min under the room temperature;
5) 4 ℃, the centrifugal 10min of 12000rpm, RNA will form precipitation in the sidewall and the bottom of pipe;
6) outwell supernatant liquor, add 75% cold washing with alcohol precipitation, 4 ℃ then, the centrifugal 5min of 12000rpm discards ethanol;
7) be deposited under the room temperature after the thorough drying, be dissolved in the 40 μ l distilled waters (DEPC processing) ,-20 ℃ of preservations are standby.
4, Klenow enzyme labelling
At first the cDNA building-up reactions promptly adds the total RNA of 10 μ L in the reaction tubes of 0.2mL, and 9N random primer (invitrogen) is 2 μ L (10mg/mL), in 70 ℃ of heating 10min, transfers to quenching 5min on ice rapidly behind the mixing, and is instantaneous centrifugal; In reaction tubes, add dNTP Mix (10mmol/L) 4 μ L then, 5 * reverse transcription damping fluid, 2 μ L, RNA enzyme inhibitors (40U/ μ L) 1 μ L, ThermoScript II (200U/ μ L) 1 μ L, mixing is instantaneous centrifugal gently; 42 ℃ of reaction 2hr, quenching 5min obtains reverse transcription product cDNA on ice.
Klenow enzyme labelling system is 25 μ L, promptly adds cDNA product 14.3 μ L in the PCR of 0.2mL reaction tubes, and 9N random primer (invitrogen) is 4 μ L (10mg/mL), 95 ℃ of sex change 3min, ice bath 5min.In reaction tubes, add 10 * Klenow enzyme buffer liquid, 2.5 μ L then, dNTP (2.5mmol/L) 2.5 μ L, cy3-dCTP 0.5 μ L, klenow enzyme (5U/ μ L) 1.2 μ L.37 ℃ of reaction 1.5hr, 70 ℃ of sex change 5min, ice bath 5min.
5, hybridization
Hybridization solution is 2.4 μ L, 3 * SSC, 0.32 μ L 0.2%SDS, 4 μ L, 25% methane amide, 0.32 μ L external standard, 1.6 μ L 5 * Denhard ' s, cy3-dCTP 0.5 μ L, mark sample 7.36 μ L.95 ℃ of sex change 3min, ice bath 5min is instantaneous centrifugal.Hybridization solution is added on the chip, and the lid thin slice is built, and 42 ℃ of water-bath hybridization are spent the night.
6, washing, scanning
Cleaning system and program are as follows:
Washing lotion I washing lotion II
Washing lotion component (ultimate density) 2 * SSC, 0.2%SDS 0.2 * SSC
42 ℃ 42 ℃ of cleaning temperatures
Scavenging period 4min 4min
Earlier washing lotion I, II are placed on and are preheating to 42 ℃ in the microwave oven, transfer in the cleaning box.Hybridization is transferred to chip in the cleaning box that holds washing lotion after finishing, and hybridization surface upwards is placed on the horizontal shaking table and slowly cleans.After chip cleans, be placed in the 50ml taper centrifuge tube, the centrifugal 1min of 2000rpm removes the liquid of chip surface.
Place scanner to carry out scanning analysis said chip, the Cy3 mark scans with the 532nm laser tube.PMT is made as 900, obtains data such as each point fluorescence intensity, background intensity.
7, data analysis
1) use LuxScan 3.0 extraction data from the chip of scanning of rich biological exploitation difficult to understand, the signal value of probe is the median of median subtracting background value of the prospect value of probe.
2) according to the standard of probe design step 6), all probes are repartitioned, probe is referred to the single virus sequence according to the virus sequence of probe correspondence.
3) corresponding all the probe signals values of each virus sequence are carried out the T check, whether the inspecting standard mean value of corresponding all the probe signals values of virus sequence for this reason uses pvalue 0.05 as screening criteria greater than 1500.So the T check pvalue of virus kind is smaller or equal to 0.05, and then this virus is planted and is positive in test sample, and Tobamovirus is positive under this virus kind.
Two, Cucumovirus virus examination chip validation verification
Be template with the Nicotiana glutinosa of representation system flower leaf paresthesia, the total RNA of burley tobaccos blade of the mottled shrinkage symptom of performance blade respectively, according to the method in () carry out that cDNA is synthetic, Klenow enzyme labelling and hybridization, can reaction finishes the back to be washed, scans chip, according to the positive hybridization signal result of determination occurring.
The hybridization hybrid chip scanning result is shown in Fig. 2 and 3, chip fixedly positive quality control, hybridization positive quality control, the negative Quality Control of hybridization and blank Quality Control performance is good, standard poison source hybridization signal does not have background noise by force, illustrates that the chip examination program of designed probe and foundation has the good operation effect.
The probe of Fig. 2 is arranged identical with Fig. 1, and positive probe is: 1,2,3,4,7,9,10,11,14 and 16.
The probe of Fig. 3 is arranged identical with Fig. 1, and positive probe is: 2,4,6,9,11,12,13,14 and 16.
Three, Cucumovirus virus examination chip and PCR detection sensitivity are relatively
The morbidity burley tobaccos blade that accurately takes by weighing 0.1g artificial inoculation tomato aspermy virus (Tomato aspcrmy virus) is used for Cucumovirus virus examination chip and PCR experiment, carries out PCR respectively and detects and chip detection.The cDNA that Cucumovirus virus examination chip is used carries out 10
1～10
9Times gradient dilution, the cDNA that PCR uses carries out 10
1～10
6Times gradient dilution.
Experimental result is shown in Figure 4 and 5, and the highly diluted multiple that PCR method can detect object to be checked is 10
5, and Cucumovirus virus examination chip can to detect the highly diluted multiple of object to be checked be 10
8Cucumovirus virus examination chip detection remolding sensitivity PCR method is high 3 times.
The probe of Fig. 4 is arranged identical with Fig. 1, A:10
-6Dilution; B:10
-7Dilution; C:10
-8Dilution.
Among Fig. 5,1:10
-1Dilution; 2:10
-2Dilution; 3:10
-3Dilution; 4:10
-4Dilution; 5:10
-5Dilution; 6:10
-6Dilution; N: blank; M:Marker DL2000.
Sequence table
＜110〉China Inst. of Quarantine Inspection Sciences
＜120〉chip of screening viruses of cucumovirus and application thereof
<160>16
<210>1
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>1
tgtagtaact?ggcaaccgaa?gttttcttcg?aagaataata?attacaagcc?agccgccgca 60
ggtaagactc 70
<210>2
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>2
ggtgaacggg?ttgtccatcc?agctaacggc?taaaatggtc?agtcgtggag?aaatccacgc 60
cagtagactt 70
<210>3
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>3
ggtctgcggc?aggacctcgt?cttagacttc?ggaggaagtt?gggtcacaca?ttacctccgc 60
ggacataacg 70
<210>4
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>4
agagagcgtg?atctcctttt?cgcttccggc?gtggataccg?gcgagagtgt?cccgagctct 60
cctgacattt 70
<210>5
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>5
ggtttcttcg?gaaggacttc?ggtccgtgta?cttctagcac?aacgtgctag?tttcagggta 60
cgggtgcccc 70
<210>6
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>6
agccgccgca?ggtaagactc?tagtactacc?ctatgacaga?ttatctgagc?tttcgggacc 60
atcagccgta 70
<210>7
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>7
tcctgaacta?tcaggttcag?ttgatttaac?tcgtatcagc?aaggccgtgg?ctagaagatt 60
taaggagtca 70
<210>8
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>8
aagtacgacc?tccatcttac?ccagcaggag?tttgctcccc?acggcctagc?tggtgccctc 60
cgcttgtgtg 70
<210>9
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>9
caagcatcga?gatccctcta?cacgagatca?ttcgaaagtt?ggaacggatg?aatcaaaaga 60
aacaagcaca 70
<210>10
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>10
accggctgcg?gattgcaaag?tacaagccca?ctttgctata?tctattcatg?gaggttatga 60
tatgggcttt 70
<210>11
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>11
ttcagatgag?ggtaccactc?tgtgtcgctt?tgacacattt?ggttccaagt?ctgatgctat 60
ttgtgatcgc 70
<210>12
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>12
cacgccagta?gacttacaag?tctctgaggc?acctttgaaa?ccatctccta?ggtttcttcg 60
gaaggacttc 70
<210>13
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>13
gtaattccta?tggcgacgtc?ctcgttcaac?atcaatgaat?tggtagcctc?ccacggcgat 60
aaaggactac 70
<210>14
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>14
caagtcggac?tctaacacaa?cagtcctctg?cggcatcaac?tgatgagcta?cataacatat 60
tatttagccg 70
<210>15
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>15
ctttccacgc?taaagggatc?acgtctcaat?tttctccttt?gtttatttcc?cttgttgaac 60
gatttcaacg 70
<210>16
<211>70
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>16
agcaattaca?gcatcaacgt?agaggccgta?aggtctacat?ccggaacgtt?ttgggtgtaa 60
aggattccga 70
Claims (4)
1. a gene chip is a DNA chip of fixing 16 kinds of dna fragmentation arrays at carrier surface, and the nucleotide sequence of described 16 kinds of dna fragmentation arrays is respectively sequence 1 in the sequence table to sequence 16.
2. the application of the described gene chip of claim 1 in the quarantine of Cucumovirus virus.
3. the method for a screening viruses of cucumovirus is to carry out Cucumovirus virus in the detection of expression plant with the described chip of claim 1.
4. the test kit of a screening viruses of cucumovirus is characterized in that: contain the described gene chip of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2009100865462A CN101570799B (en) | 2009-06-05 | 2009-06-05 | Chip for screening viruses of cucumovirus and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100865462A CN101570799B (en) | 2009-06-05 | 2009-06-05 | Chip for screening viruses of cucumovirus and application thereof |
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| Publication Number | Publication Date |
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| CN101570799A CN101570799A (en) | 2009-11-04 |
| CN101570799B true CN101570799B (en) | 2011-10-05 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108342508A (en) * | 2017-12-01 | 2018-07-31 | 贵州省果树科学研究所 | A kind of passionflower mosaic virus early detection method |
| CN112331268B (en) * | 2020-10-19 | 2023-04-14 | 成都基因坊科技有限公司 | Method for obtaining specific sequence of target species and method for detecting target species |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
| CN101186950A (en) * | 2007-06-07 | 2008-05-28 | 中国检验检疫科学研究院 | Primer and probe for detecting cucumber green mottle mosaic virus |
-
2009
- 2009-06-05 CN CN2009100865462A patent/CN101570799B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952648A (en) * | 2005-10-19 | 2007-04-25 | 中华人民共和国北京出入境检验检疫局 | A gene chip, nucleotide sequence and reagent kit for detecting virus of leguminous plant |
| CN101186950A (en) * | 2007-06-07 | 2008-05-28 | 中国检验检疫科学研究院 | Primer and probe for detecting cucumber green mottle mosaic virus |
Non-Patent Citations (1)
| Title |
|---|
| Zhang Deyong et al..Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format.《Journal of Virological Methods》.2005,第123卷 * |
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Inventor after: Zhang Yongjiang Inventor after: Zhu Shuifang Inventor after: Yin Jun Inventor after: Huang Xin Inventor after: Li Mingfu Inventor after: Li Guifen Inventor before: Zhang Yongjiang Inventor before: Zhu Shuifang Inventor before: Huang Xin Inventor before: Li Mingfu Inventor before: Li Guifen |
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Free format text: CORRECT: INVENTOR; FROM: ZHANG YONGJIANG ZHU SHUIFANG HUANG XIN LI MINGFU LI GUIFEN TO: ZHANG YONGJIANG ZHU SHUIFANG YIN JUN HUANG XIN LI MINGFU LI GUIFEN |
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Effective date of registration: 20130710 Address after: 100025 No. 3, Gaobeidian North Road, Beijing, Chaoyang District Patentee after: Chinese Academy of Inspection and Quarantine Patentee after: Yin Jun Address before: 100025 No. 3, Gaobeidian North Road, Beijing, Chaoyang District Patentee before: Chinese Academy of Inspection and Quarantine |
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Granted publication date: 20111005 Termination date: 20140605 |