CN101705309B - Kit for auxiliarily detecting tobacco ringspot virus and application thereof - Google Patents
Kit for auxiliarily detecting tobacco ringspot virus and application thereof Download PDFInfo
- Publication number
- CN101705309B CN101705309B CN2009102416345A CN200910241634A CN101705309B CN 101705309 B CN101705309 B CN 101705309B CN 2009102416345 A CN2009102416345 A CN 2009102416345A CN 200910241634 A CN200910241634 A CN 200910241634A CN 101705309 B CN101705309 B CN 101705309B
- Authority
- CN
- China
- Prior art keywords
- sequence
- virus
- tobacco ringspot
- kit
- ringspot virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for auxiliarily detecting tobacco ringspot virus and an application thereof. The kit comprises a specific primer pair and/or a specific probe; the specific primer pair is a primer pair formed by nucleotide sequences shown in a sequence 1 and a sequence 2 in a sequence table; and the nucleotide sequences of the specific probe are shown in a sequence 3 of the sequence table. The kit can be applied to detecting the tobacco ringspot virus, can detect the tobacco ringspot virus in a fast, accurate and quantitative manner, can judge whether a sample contains virus according to whether a fluorescence signal is generated and can judge the content of the virus in the sample according to the size of the generated fluorescence signal value. The kit has the advantages of strong specificity, high accuracy, simple and convenient detection process operation (detection can be carried out by only one real-time fluorescence PCR instrument and electrophoresis does not need to be carried out), short time needed by detection (only three hours are needed from the extraction of the tobacco ringspot virus to the occurrence of detection result) and the like, and is suitable for applications of all quarantine inspection stations in agriculture sectors, all port inspection quarantine bureaus, plant virus research institutions and the like.
Description
Technical field
The present invention relates to a kind of test kit and application thereof of auxiliarily detecting tobacco ringspot virus.
Background technology
TRSV (Tobacco ring spot virus) is the member of Comoviridae Nepovirus.Virus particle is for waiting axle polyhedron, and diameter is about 28nm.Under the natural condition, the TRSV host range is wide, can infect 54 sections, 246 kind of plant.Various because of its route of transmission, harm is serious and classified as Quarantine Objects by many countries in the world.At present, nepovirus all has distribution in more than 50 countries such as the U.S., India, Indonesia, Japan, China, Iran, Britain, Bulgaria, Czech, Denmark, France, Germany, Greece, Hungary, Italy, Holland.The country that classifies nepovirus as Quarantine Objects in the world has: the FSU, Germany, Australia, Canada, South Africa, nz, Israel, Chile, Malta, East Africa etc.Cause the soybean yields loss more than 50% in China, but the Kidney bean underproduction 30～50% is classified TRSV as two types of Quarantine Objectss in the inward Quarantine Objects list of announcement in 1992.Setting up fast and accurately, virus detection techniques has important practical significance to ensureing crop safety production and the venereal disease poison diffusion that prevents to quarantine.
At present, generally use ELISA both at home and abroad, colloidal gold immunity chromatography, regular-PCR, multiplex PCR, methods such as immunocapture PCR detect TRSV, but the limitation that there is false positive results in these methods and is difficult to carry out the copy number detection of starting template.This area press for high specificity, highly sensitive, convenient, detect primer and method accurately.
The real-time fluorescence PCR technology is in vitro accomplished pcr amplification, fluorogenic probe hybridzation and signal detection at same PCR, reduces possibility of pollution, improves detection specificity, can carry out quantitatively pathogen, thereby overcome above-mentioned limitation.
Summary of the invention
The purpose of this invention is to provide a kind of test kit and application thereof of auxiliarily detecting tobacco ringspot virus.
The test kit of auxiliarily detecting tobacco ringspot virus provided by the invention, comprise special primer to and/or specific probe; Said special primer is right to the primer of forming for nucleotide sequence shown in the sequence 2 of sequence of sequence table 1 and sequence table; The nucleotide sequence of said specific probe is shown in the sequence 3 of sequence table.
Special primer is to as follows:
Upstream primer (F): 5 '-GAGTTTATTGTACATATAGATACCTGGCG-3 ' (sequence 1 of sequence table);
Downstream primer (R): 5 '-CGAAGTCATGAATGTATCCAGG-3 ' (sequence 2 of sequence table).
Specific probe (TaqMan probe) (nucleotides sequence is classified the sequence 3 of sequence table as) is as follows:
5’-FAM-TGACTCTCAGGTGCATCCTCCCATGTT-TAMRA-3’。
Said test kit can also comprise that the conventional reagent of other PCR, RNA extract reagent and the conventional reagent of reverse transcription etc.
The application of said test kit in detecting nepovirus also belongs to protection scope of the present invention.
The present invention also protects a kind of special primer of auxiliarily detecting tobacco ringspot virus right, for the primer that nucleotide sequence shown in the sequence 2 of sequence of sequence table 1 and sequence table is formed right.
The present invention also protects a kind of specific probe of auxiliarily detecting tobacco ringspot virus, and the nucleotide sequence of said specific probe is shown in the sequence 3 of sequence table.Said probe specifically can be the TaqMan probe.
Said special primer to and/or said specific probe can be applicable to prepare the test kit of auxiliarily detecting tobacco ringspot virus.
Said special primer to and/or said specific probe can be applicable at auxiliarily detecting tobacco ringspot virus.
The present invention also protects a kind of method that whether contains nepovirus in the sample to be tested that detects, and comprises the steps: that the cDNA with sample to be tested is a template, to carrying out PCR, detects the PCR product with special primer; If obtain the PCR product of 165bp, then contain nepovirus in the sample to be tested; If do not obtain the PCR product of 165bp, then do not contain nepovirus in the sample to be tested; Said special primer is right to the primer of forming for nucleotide sequence shown in the sequence 2 of sequence of sequence table 1 and sequence table.
Said PCR specifically can be real-time fluorescence quantitative PCR; Used probe can be the TaqMan probe in the said real-time fluorescence quantitative PCR; The nucleotide sequence of said TaqMan probe specifically can be shown in the sequence 3 of sequence table.
Adopt test kit provided by the invention; Carrying out real-time fluorescence PCR with primer pair and specificity T aqMan probe detects; According to whether producing fluorescent signal; Can whether contain nepovirus in the judgement sample,, get final product the height of nepovirus content in the judgement sample according to the size that produces the fluorescent signal value.
Compare with prior art, advantage of the present invention is following:
1) the detected result accuracy is high.In the process of exploitation and check special primer pair and specific probe, 96 samples have been verified altogether, to the detection rate of accuracy reached of nepovirus more than 95% from a plurality of provinces of the U.S. and China.
2) detection sensitivity is high.At least can detect the viral RNA of 767 copies.
3) can detection by quantitative.On the basis of qualitative detection nepovirus, can obtain the accurate content of virus in the sample.
4) detection method safety.Need not carry out electrophoresis detection, avoid the pyridine of bromination second and some dyestuffs the pollution of environment and the damage of human body.
5) detected result is easy to interpretation.Carry out real-time fluorescence PCR with primer pair and specificity T aqMan probe and detect,, can whether contain nepovirus in the judgement sample according to whether producing fluorescent signal; According to the size that produces the fluorescent signal value, get final product the height of nepovirus content in the judgement sample.
6) detecting instrument is easy.Only need the real-time fluorescence PCR appearance, need not electrophoresis apparatus.Being adapted at Check and Examination of Port quarantine office promotes.
The invention discloses a kind of contain special primer to the test kit of specific probe, can detect nepovirus fast, accurately, stably, quantitatively.According to whether producing fluorescent signal, can whether contain nepovirus in the judgement sample, according to the size that produces the fluorescent signal value, get final product the height of nepovirus content in the judgement sample.Test kit of the present invention has high specificity, accuracy is high, testing process is easy and simple to handle (only needs a real-time fluorescence PCR appearance to detect; Need not to carry out the electrophoresis operation), detect the short advantages such as (occurring only needing 3 hours) of required time from the detected result of extracting of nepovirus, be fit to the application such as each Quarantine Check station, each Check and Examination of Port quarantine office, plant virus research institution of agricultural sector.
Description of drawings
Fig. 1 is the sensitivity and the repeatability of TRSV fluorescent quantitation; 1-6 is followed successively by: 7.67 * 10
7, 7.67 * 10
6, 7.67 * 10
5, 7.67 * 10
4, 7.67 * 10
3, 7.67 * 10
2
Fig. 2 is the specificity of TRSV fluorescent quantitation.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.
The real-time fluorescence PCR appearance is an ABI7300PCR amplification appearance (ABI company).Test used ReverseTranscription System A3500 and dNTPs and purchase company in Promega.Plant virus nucleic acid nanometer magnetic bead rapid extraction test kit is believed company available from Beijing Jenner.Taq enzyme, ligase enzyme, PMD18-T and DNA MarkerDL2000 are available from TaKaRa.The real-time fluorescence PCR agents useful for same is available from ABI company.Primer and probe are synthetic by the precious biotech company in Dalian.
Mouseearcress mosaic virus (ArMV): ATCC (USS type culture collection institute); Catalog number: PV-192.
Potato virus X (PVX): ATCC (USS type culture collection institute); Catalog number: PV-197.
Marmor upsilon (PVY): ATCC (USS type culture collection institute); Catalog number: PV-50.
Nepovirus (TRSV): ATCC (USS type culture collection institute); Catalog number: PV-97.
The preparation of embodiment 1, test kit
Test kit by special primer to forming with specific probe.
Special primer is to as follows:
Upstream primer (F): 5 '-GAGTTTATTGTACATATAGATACCTGGCG-3 ' (sequence 1 of sequence table);
Downstream primer (R): 5 '-CGAAGTCATGAATGTATCCAGG-3 ' (sequence 2 of sequence table).
Specific probe (TaqMan probe) (nucleotides sequence is classified the sequence 3 of sequence table as) is as follows:
5’-FAM-TGACTCTCAGGTGCATCCTCCCATGTT-TAMRA-3’。
Carry out the mensuration of embodiment 2 and embodiment 3 with the test kit of embodiment 1.
The susceptibility of embodiment 2, test kit and repeatability are measured
1, the extraction of viral RNA
Take by weighing this lifes of 0.1g morbidity tobacco leaf sheet (design specific primers, and through sequence verification, confirm to contain nepovirus does not contain other tobacco virus), adopt plant virus nucleic acid nanometer magnetic bead rapid extraction test kit extraction viral RNA.Measure its concentration with NanoDrop ND-1000Spectrophotometer quantitative analysis instrument.
The Auele Specific Primer of checking nepovirus is following:
TRS?V-F：5’-ATGGGTGCTGTGACAGTTGTTC-3’
TRSV-R：5’-GGACAAACACGACACTAGGAAAC-3’
Sequencing result is seen the sequence 4 of sequence table.
2, cDNA's is synthetic
When viral RNA being carried out 10 times of concentration gradient dilutions, each gradient repeats 3 times.
Synthetic system (20.0 μ L):
DEPC-H
2O 7.8μL
10×buffer 2.0μL
MgCl
2(25mmol/L) 4.0μL
dNTP(10mmol/L) 1.0μL
Random hexamer primer 2 .0 μ L
RNasin(44U/μL) 0.5μL
Viral RNA 2.0 μ L
AMV?Reverse?Transcriptase(22U/μL)0.7μL
42 ℃ of water-bath 1h behind the synthetic system mixing, 95 ℃ of deactivation 5min place for use on ice.
3, real-time fluorescence PCR amplification
The real-time fluorescence PCR reaction system: 2 * Master Mix, 12.5 μ L, each 2 μ L of 10mmol/L upstream and downstream primer, 10mmol/L specific probe 1 μ L adds dd H
2O to TV be 25 μ L.
PCR program: 50 ℃ of 5min; 95 ℃ of 10min; 95 ℃ of 15s, 60 ℃ of 1min circulate 40 times.
The result is as shown in Figure 1.This method stability is strong, and good reproducibility is highly sensitive, can detect 767 virus copies at least.
The specific assay of embodiment 3, test kit
CDNA with four kinds of viruses (ArMV, PVX, PVY and TRSV) is a template, the real-time fluorescence PCR amplification, and method is with embodiment 2.
The result sees Fig. 2.The result shows that have only TRSV typical amplification curve to occur, Ct value reading is 21.5, is judged to the positive, and ArMV, the amplification of PVX and PVY is smooth straight line, is judged to feminine gender.Thus it is clear that, adopt test kit of the present invention to detect and have very strong detection specificity.
Sequence table
< 110>China Inst. of Quarantine Inspection Sciences
< 120>a kind of test kit of auxiliarily detecting tobacco ringspot virus and application thereof
<130>CGGNARY92722
<160>4
<210>1
<211>29
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>1
gagtttattg?tacatataga?tacctggcg 29
<210>2
<211>22
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
cgaagtcatg?aatgtatcca?gg 22
<210>3
<211>27
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
tgactctcag?gtgcatcctc?ccatgtt 27
<210>4
<211>2050
<212>DNA
< 213>nepovirus (Tobacco ringspot virus)
<400>4
gacaaacacg?acactaggaa?acataaaagg?ccccaaacca?aatatgaaca?gtgggctcaa 60
acaacagcaa?atagcaaagc?agctttagaa?aactcaatag?acccaacaaa?gctgccagaa 120
acaacggtct?aacacatttt?gttaactaga?gattttactc?cggtcgtctg?gctactaact 180
tgccacgaca?gtagttttca?cttgtgccca?ggagagctat?cctgggagaa?agctcattta 240
cacaagcagc?tgacagacag?acattcttgg?gctctcaccc?ccaagaagct?gagtttatac 300
agcttcccga?tccacctaag?gaccggacca?ccctagtaag?ggcaggaacc?taaaacgaaa 360
ctaattaaag?tcccggttta?ttgacgcttc?tatctaacca?accatgtaca?gcatggttac 420
cagcaaacta?atgccagcag?ggctaccaat?cctggttaga?agaatcaaaa?gacacaacac 480
ctaattaaag?gcatttaaaa?caaagtggcg?gagcggcccc?agaactttat?agatttgggt 540
cgcaaacaca?ctcgcaatgc?aactatgtcc?ttagcattga?caattgaaat?tcgaacatag 600
ttttgcttac?ccaaagcacc?agatggcgtc?gcaccagcat?aagaacccaa?gaaaatgggg 660
atttccgcct?cggtgcgccg?caaatcataa?atccgagcct?ggccttgcca?ataagcaccc 720
caatctctat?aattggtggt?gatggaaaca?ctgccctcct?gagcctgggc?ctgtttagac 780
cttgaccaag?aaatgcagag?atccacctca?ccataatgcc?agccagtggc?tgcaacaagc 840
caagaaaggg?gattcgatgc?gagcgtcaca?gttgcgtctt?tagaggcgaa?gtcatgaatg 900
tatccaggga?tatcaaaatt?gaccacgcca?gtgttcgcat?ccggtttcaa?attggttaaa 960
gtaaaccaat?tataaagctc?cgcactagaa?aacatgggag?gatgcacctg?agagtcgggt 1020
cgccaggtat?ctatatgtac?aataaactcg?cacgacccaa?catgtgcatc?aggagctatg 1080
ggaccagaaa?gagggagaat?gccaagtcta?ccaaatcctt?cttcagttgt?agaagatgaa 1140
tgaagacggg?tcttggtagc?aatttcaaac?tggccatctc?cgtgcattat?ctgatgtggg 1200
ccccacaaca?cctgacgcaa?agtcggaggt?gtttcatgtt?cccatgggaa?aataacaaaa 1260
gaagcggtac?aaaataaaga?agaaacactc?ttgacacttc?cgcttatagt?gccagaccat 1320
ccaagattcc?tggacataac?tgcccgagaa?tatgaaatgg?catgcttctt?agaatgctta 1380
acagggcctc?cgaaattata?agatatggac?aacatggagg?tcgacccatc?aagctttatt 1440
tctttcgcct?cataatatct?atcaatggcg?aaagtgtcac?ccaattgaat?gggccacgtc 1500
atagttggat?ctaactcaaa?tcgatcaatg?acgggcccca?tgtccacatg?catagtaaca 1560
atacactgcc?aatcagcagc?catcgtcacc?tgatttgtgg?atgcaacaaa?aaagtacaat 1620
gtgggctggg?taactacatt?tgcccaattg?cacaacccat?gtcctgtaag?ttccccatag 1680
ttgaactgcc?actcatgcac?atctgcatct?ttaagcatga?aaaccttggt?aggcagctca 1740
aacatctcag?agggaggaca?ttcattccca?agcgctggta?agtctatgcg?tttataatca 1800
tcaaaggtgc?acgctataga?caaacctgca?aaggcgttgc?gtggggcatg?catcctcact 1860
gtaaaggtgg?gattaacaat?gccctttgca?caccattctt?gggaatgcaa?accaccataa 1920
tccataatgg?cttgccgaat?gtcaaaggtt?ccaagatgct?ttcctttctt?tgcatctttg 1980
ggaactttaa?aggacaatgt?cccacaacaa?gtgggatcag?gaacaactgt?cacagcaccc 2040
ataatctcta 2050
Claims (6)
1. the test kit of an auxiliarily detecting tobacco ringspot virus, comprise special primer to and/or specific probe; Said special primer is right to the primer of forming for nucleotide sequence shown in the sequence 2 of sequence of sequence table 1 and sequence table; The nucleotide sequence of said specific probe is shown in the sequence 3 of sequence table.
2. the application of the said test kit of claim 1 in detecting nepovirus.
3. the special primer of an auxiliarily detecting tobacco ringspot virus is right, for the primer that nucleotide sequence shown in the sequence 2 of sequence of sequence table 1 and sequence table is formed right.
4. the specific probe of an auxiliarily detecting tobacco ringspot virus, the nucleotide sequence of said specific probe is shown in the sequence 3 of sequence table.
The said special primer of claim 3 to and/or the said specific probe of claim 4 the preparation auxiliarily detecting tobacco ringspot virus test kit in application.
The said special primer of claim 3 to and/or the application of the said specific probe of claim 4 in auxiliarily detecting tobacco ringspot virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102416345A CN101705309B (en) | 2009-12-04 | 2009-12-04 | Kit for auxiliarily detecting tobacco ringspot virus and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102416345A CN101705309B (en) | 2009-12-04 | 2009-12-04 | Kit for auxiliarily detecting tobacco ringspot virus and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101705309A CN101705309A (en) | 2010-05-12 |
| CN101705309B true CN101705309B (en) | 2012-04-18 |
Family
ID=42375561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102416345A Expired - Fee Related CN101705309B (en) | 2009-12-04 | 2009-12-04 | Kit for auxiliarily detecting tobacco ringspot virus and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101705309B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102230021B (en) * | 2011-06-03 | 2013-04-03 | 宁波检验检疫科学技术研究院 | Tobacco ringspot virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection reagent and preparation method and use thereof |
| CN102277450B (en) * | 2011-08-16 | 2013-04-03 | 厦门出入境检验检疫局检验检疫技术中心 | Primer used for multiple detection of five quarantine nepoviruses and application thereof |
| CN109988858B (en) * | 2017-12-29 | 2023-09-22 | 贵州中烟工业有限责任公司 | Multiple fluorescence PCR gene locus, primer and detection method of transgenic tobacco |
| CN113215319A (en) * | 2021-05-21 | 2021-08-06 | 中国计量大学 | Cross primer isothermal amplification primer group for detecting tobacco ringspot virus, kit and application |
-
2009
- 2009-12-04 CN CN2009102416345A patent/CN101705309B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 杨伟东等.烟草环斑病毒IC_RT_RealtimePCR检测方法研究.《中国病毒学》.2006,第21卷(第3期),第277-280页. * |
| 杨伟东等.烟草环斑病毒RT—Realtime PCR检测方法.《植物保护学报》.2007,第34卷(第2期),第157-160页. * |
| 郑耘等.烟草环斑病毒DB_RT_RealtimePCR检测方法研究.《植物保护》.2007,第33卷(第1期),第117-120页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101705309A (en) | 2010-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Roy et al. | A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees | |
| KR101692436B1 (en) | A primer set for diagnosing Zika virus, a kit for diagnosing Zika virus comprising the same, and a method of diagnosing Zika virus using the same | |
| CN101705309B (en) | Kit for auxiliarily detecting tobacco ringspot virus and application thereof | |
| Shang et al. | Development and validation of an efficient in-house real-time reverse transcription polymerase chain reaction assay for the quantitative detection of serum hepatitis delta virus RNA in a diverse South London population | |
| CN101956021B (en) | Composition, kit and method used for detecting enterovirus causing hand foot and mouth disease | |
| Xiao et al. | Comparative evaluation of a triplex nucleic acid test for detection of HBV DNA, HCV RNA, and HIV-1 RNA, with the Procleix Tigris System | |
| CN102827950A (en) | Fluorescence PCR kit for rapidly quantitative detection of gene C-type duck hepatitis A virus | |
| CN109161611B (en) | Primer probe set and kit for jointly detecting banna virus, Canadenuovirus and Liaoning virus based on triple fluorescence PCR method | |
| CN103937908B (en) | The real-time fluorescence quantitative PCR detection method of sweet potato chlorotic stunt virus West Africa strain and application | |
| CN105368943A (en) | Kit and method for identifying mycobacterium strains | |
| Xia et al. | Rapid detection of Banna virus by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) | |
| CN107828858A (en) | A kind of method that exploitation beggar-ticks plant SSR primers are sequenced based on transcript profile | |
| CN108950080B (en) | Primer probe set and kit for jointly detecting Sindbis virus and Getavirus based on dual-fluorescence PCR method | |
| Jiang et al. | Conventional octaplex PCR for the simultaneous identification of eight mainstream closely related Dendrobium species | |
| CN102031314A (en) | Primer and probe sequence for human metapneumovirus nucleonic acid detection | |
| Jiang et al. | CRISPR Cas12a-mediated amplification-free digital DNA assay improves the diagnosis and surveillance of Nasopharyngeal carcinoma | |
| WO2003054214A3 (en) | Tsunami chain reaction - geometric dna amplification | |
| CN103805717A (en) | Dual Eva Green real-time fluorescence quantification PCR (Polymerase Chain Reaction) detection kit for detecting equine herpesviruses I and IV and application thereof | |
| KR101024114B1 (en) | PCR primer pair detecting Pea enation mosaic virus and method for detecting Pea enation mosaic virus using the same | |
| CN107385048A (en) | A kind of Nucleic acid combinations, kit and method for detecting dendrobium candidum | |
| KR102173154B1 (en) | Loop Mediated Isothermal Amplification Primer Set for Detection of Dengue Virus Serotype 1 or 3 and Uses Thereof | |
| Guanghui et al. | A general onepot-method for nucleic acid detection with CRISPR-Cas12a | |
| CN106282389A (en) | The primer of dual nido fluorescent PCR detection genetically engineered soybean DAS44406 and probe combinations, method and test kit | |
| CN101928786B (en) | Kit for aided identification of Plum Pox virus and application thereof | |
| CN111206117A (en) | Kit for detecting human immunodeficiency virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120418 Termination date: 20171204 |