CN102031307B - Kit for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis - Google Patents
Kit for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis Download PDFInfo
- Publication number
- CN102031307B CN102031307B CN 201010550620 CN201010550620A CN102031307B CN 102031307 B CN102031307 B CN 102031307B CN 201010550620 CN201010550620 CN 201010550620 CN 201010550620 A CN201010550620 A CN 201010550620A CN 102031307 B CN102031307 B CN 102031307B
- Authority
- CN
- China
- Prior art keywords
- liquid
- pipe
- giant salamander
- ecdysis
- sample
- 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 and for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis. The kit comprises 1 tube of solution A, namely ecdysis sample lysis solution containing PBS (phosphate buffer saline) and 1% triton-100, 1 tube of solution B containing 200mug/ml of protease K, 1 tube of solution C, namely template extraction solution containing phenol, chloroform and isoamyl alcohol based on the proportion of 25:24:1, 1 tube of solution D containing 3mol/l sodium acetate and the like. In the invention, the fresh Chinese giant salamander ecdysis is taken as a pathological material detection sample, thus solving the defect that the detection sample is obtained by killing Chinese giant salamander individuals; according to the characteristics of the Chinese giant salamander ecdysis, the method for extracting virus genome DNA from the ecdysis is optimized, and the kit for simply and effectively extracting an iridovirus gene from the Chinese giant salamander ecdysis sample as well as 2 pairs of PCR (polymerase chain reaction) primers for 2 conserved genes of the iridovirus and PCR conditions are designed; and positive and negative reference substances are established. By utilizing the kit for detection in standard steps, the virus infecting situation of the Chinese giant salamander colony iridovirus can be more reliably mastered, thus being favorable for application to actual production.
Description
Technical field
The present invention relates to a kind of utilization casts off a skin and detects test kit and the method for giant salamander group irido virus band poison situation.
Background technology
The distribution of giant salamander in the whole world mainly is Japan, North America and China; aspect Resources of Megalobatrachus Davidianus protection and industry development; although Andrias japonicus and U.S. cryptobranchid in various degree to some extent attention in conservation of resources; China raises and train aspects such as breeding art research and development utilization giant salamander and has been in the first place in the world; artificially-cultured giant salamander not only becomes the means of ecological environmental protection, and has become the Special Breed industry in part area.Artificially-cultured giant salamander has the great ecological value and economic worth.
China giant salamander mainly is distributed in the mountain stream streams in the Changjiang river, the Yellow River and Zhujiang River middle and upper reaches tributary.Wild giant salamander country of origin NATURAL DISTRIBUTION mainly concentrates on five provinces of China, comprises Shaanxi, Hubei, Hunan, Guizhou and the fourth class.At present the nationwide all in various degree the giant salamander of having carried out raise and train breeding work, along with the continuous expansion of cultivation scale, various communicable diseases are outburst and popular constantly, has seriously restricted the Sustainable development of giant salamander culture.From 2009 to 2010, according to incomplete statistics, the whole nation reached more than 100,000 because of the giant salamander quantity of iridescent virus disease death, and financial loss is serious, was also endangering wild giant salamander group's health procreation simultaneously.Utilizing liver, spleen and the kidney of the methods such as electron microscope and PCR in the dead giant salamander body to detect irido virus is feasible method, but is difficult to be applied in the actual production.
What report had been arranged all is that liver and spleen from death or sick giant salamander detects, this detection method only to fall ill or dead giant salamander to detect be effective, still do not adopt this method then to cause the individual injury of giant salamander to showing clinical symptom or healthy giant salamander group, be not suitable for healthy giant salamander group's monitoring fully.Up to the present, also there is not a kind of detection method to the individual nothing injury of giant salamander.How whether efficient and convenient making a definite diagnosis exists irido virus among the giant salamander group, and is applied to not be with malicious giant salamander to select and introduce a fine variety, and reduces virus and propagates in the giant salamander group, significant to giant salamander culture and protection.
The extracting method of viral DNA and round pcr are to be widely used at present scientific research and the actual ripe means that detect.For the giant salamander genomic simply and effectively extracting method of middle irido virus of casting off a skin, and detect also report not of giant salamander irido virus band poison situation take this DNA as template PCR.
Summary of the invention
Technical problem to be solved by this invention is: technical problem to be solved by this invention is test kit and the method that provides a kind of utilization to cast off a skin detection giant salamander group irido virus band poison situation for the prior art deficiency.
The present invention is by the following technical solutions:
1. non-damage live body giant salamander sample acquisition methods
By obtaining at random fresh casting off a skin among the giant salamander group, and do not injure any one individuality of giant salamander group, overcome the problem that just can obtain sample from the viscera tissue of giant salamander.Only need that the sample of casting off a skin that will obtain is frozen can preserve or be directly used in detection the long term in-20 ℃, we use this method under study for action first, effect fine (seeing accompanying drawing and explanation).This method of obtaining sample can be with malicious situation to detect to introducing a fine variety the giant salamander group, avoids and will be with virulent giant salamander to introduce plant; Utilize simultaneously this infection conditions without injuring the sample tracking monitor giant salamander group irido virus in breeding process that obtains, in order in time take corresponding measure, reduce the loss.
2. irido virus DNA detection test kit and the detection method in the extraction sample
We have optimized the method for extraction virus genom DNA from cast off a skin the characteristics of casting off a skin according to giant salamander.By designing corresponding test kit, and utilize test kit to carry out the detection of standard step, more be conducive in actual production, use.
Test kit comprises DNA extraction agent combination and pcr amplification agent combination; Extract the viral DNA agent combination in the sample:
A liquid, the sample lysate of casting off a skin, 1 pipe mainly contains PBS; 1%triton-100;
B liquid, 1 pipe, 200ug/ml Proteinase K;
C liquid, 1 pipe, the template extract, 1 pipe contains phenol/chloroform/primary isoamyl alcohol, and ratio is 25: 24: 1;
D liquid, 1 pipe, 3mol/l sodium acetate;
E liquid, 1 pipe contains dehydrated alcohol;
F liquid, 1 pipe contains 75% ethanol;
The G pipe, 1 pipe contains the sterilization ultrapure water;
Pcr amplification pcr amplification agent combination:
H liquid, 1 pipe contains pcr amplification reaction liquid, comprises deionized water, and the dNTP substrate contains Mg
2+2 * damping fluid; The Taq enzyme; Primer (can according to primer quantity is increased the pipe number and according to primer isolabeling not);
I liquid, 1 pipe, positive control sample;
J liquid, 1 pipe, negative control product;
K liquid, 1 pipe, DNA marker II
In the box of mentioned reagent packing with cystose.Each tubule is placed in the aperture of cystose.The centrifuge tube of 100 aseptic 1.5ml and 50 PCR pipes are placed on respectively in two plastics bags, and each test kit can at least 10 samples of one-time detection.
3 usefulness mentioned reagent box of the present invention detects the giant salamander middle irido virus of casting off a skin.Undertaken by following concrete steps:
1) the giant salamander group of cultivation in same pond gets the 3-5 sheet of casting off a skin with ophthalmology tweezers and scissors at random, removes moisture as far as possible, is kept in the centrifuge tube of 2 μ l, cut 10-20 time with sampling shears simultaneously, mixing, after mark is good, be kept at-20 ℃ for subsequent use.Must notice that each culturing pool sampling apparatus can not cross-reference.
2) cast off a skin in the new 1.5ml centrifuge tube of sample 0.1g, add A liquid 500 μ l, repeatedly freeze molten 3 times, add B liquid 20 μ l, behind 55 ℃ of water-bath 1.0h, the centrifugal 10min of 4000rpm removes the fragment of casting off a skin;
3) get supernatant 500 μ l in new 1.5ml centrifuge tube, add equal-volume (500 μ l) C liquid extracting, mix, room temperature is placed 5min, the centrifugal 5min of 12000rpm;
4) get supernatant in new 1.5ml centrifuge tube, add 1/10 volume D liquid, 3 times of volume E liquid are put-20 ℃ of 30min precipitation DNA, and the centrifugal 10min of 13,000rpm carefully outwells supernatant liquor;
5) add 500 μ l F liquid in above-mentioned centrifuge tube, the centrifugal 5min of 13,000rpm carefully abandons supernatant, comes again;
6) dry rear adding 30 μ l G liquid dissolving in room temperature; Be template DNA solution to be measured.
7) sample thief quantity+2 a PCR pipe, lable number (carrying out mark according to the sample of measuring), the H liquid that adds respectively 19 μ l, and the liquid (template DNA solution to be measured), I liquid and the J liquid 1 μ l that got respectively for the 6th step join in the PCR pipe of correspondence markings number, after being mixed, the centrifugal 30sec of 5000rpm places on the PCR instrument.
Increase by following condition:
95 ℃ of sex change 5min → 95 ℃, 30sec;
58℃，30sec；
72 ℃, 30sec (30 circulations) → 72 ℃, 10min → 4 ℃ preservation.
8) after reaction finished, the tetrabromophenol sulfonphthalein that adds 4 μ l in each PCR was mixed, and the sepharose of preparation 1% adds respectively the mixed solution of 15 μ l in the well, added the DNA marker II of 8 μ l in another hole, 120V electrophoresis 20min.
9) after the result judges that electrophoresis finishes, observe under ultraviolet lamp, the contrast positive and negative control if sample has band to show that this corresponding giant salamander group infects irido virus at identical molecular weight place, otherwise do not infect.
The present invention utilizes the fresh of giant salamander to cast off a skin as detecting samples first, has solved and must kill the deficiency that the giant salamander individuality just can obtain test sample; After the optimization, designed and simply and effectively extracted the cast off a skin test kit of the virogene in the sample of giant salamander; The 2 pairs of PCR primers and polymerase chain reaction system and condition for 2 conservative genes of irido virus; Set up reliability positive and negative control product control detected result.Utilize test kit of the present invention and standard detecting method, more be adapted in the reality of giant salamander culture production, use.
Description of drawings
Fig. 1 is the electrophoresis photo.M:DNA Marker; 1 swimming lane: positive control; 2 swimming lanes: negative control; 3-12 swimming lane: be respectively ten different samples; S1: primer S1; S2: primer S2.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
A giant salamander irido virus detection kit trial-production
(specification: detect 10 samples by a test kit)
A liquid, the sample lysate of casting off a skin, 1 pipe 12ml mainly contains PBS; 1%triton-100
B liquid, 1 pipe 400ul, 200ug/ml Proteinase K;
C liquid, 1 pipe, the template extract, 1 pipe 12ml contains phenol/chloroform/primary isoamyl alcohol, and ratio is 25: 24: 1;
D liquid, 1 pipe 2ml, 3mol/l sodium acetate;
E liquid, 1 pipe 20ml contains dehydrated alcohol;
F liquid, 1 pipe 40ml contains 75% ethanol;
The G pipe, 1 pipe 1ml contains the sterilization ultrapure water;
H liquid, S1 are managed 300 μ l, contain pcr amplification reaction liquid (20 μ l system), comprise deionized water, and the dNTP substrate contains Mg
2+1 * damping fluid; The Taq enzyme; Primer S1 (5 '-CCCCTCCCATTCTTCTTCTCC-3 ' and
5’-GGCGTTGGTCAGTCTACCGTAAT-3’)。
S2 manages 300 μ l, contains pcr amplification reaction liquid (20 μ l system), comprises deionized water, and the dNTP substrate contains Mg
2+1 * damping fluid; The Taq enzyme; Primer S2 (5 '-GGGCTAATGTATTGAAGACGC-3 ' and 5 '-TTGTAAACTTGGAGTGGAGGG-3 '),
I liquid, 1 pipe, 3 μ l positive control sample; Contain the irido virus dna profiling
J liquid, 1 pipe, 3 μ l negative control product contain cow genome group DNA mixed solution
K liquid, 1 pipe, 30 μ l, DNA marker II
(length * wide * height=15 * 15 * 15cm in the carton box of mentioned reagent packing with cystose
3).Each tubule is placed in the aperture of cystose.The centrifuge tube of 100 aseptic 1.5ml and 30 PCR pipes are placed on respectively in two plastics bags.
Each test kit can at least 10 samples of one-time detection.
Embodiment 2:
Detect 10 giant salamander group irido virus band poison situations with mentioned reagent box of the present invention:
1. by requirement of the present invention, obtain different 10 giant salamander groups and cast off a skin each 5, in the centrifuge tube of 2 μ l, cut 10-20 time respectively mixing with sampling shears.
2. in the new 1.5ml centrifuge tube of (3-12 number) of the sample 0.1g number of finishing of casting off a skin, add A liquid 500 μ l, repeatedly freeze molten 3 times, add B liquid 20 μ l, behind 55 ℃ of water-bath 1.0h, the centrifugal 10min of 4000rpm removes the fragment of casting off a skin.
3. get supernatant 500 μ l (step numbering on the correspondence) in new 1.5ml centrifuge tube, add equal-volume (500 μ l) C liquid extracting, mix, room temperature is placed 5min, the centrifugal 5min of 12000rpm;
4. get supernatant (step numbering on the correspondence) in new 1.5ml centrifuge tube, add 1/10 volume D liquid, 3 times of volume E liquid are put-20 ℃ of 30min precipitation DNA, and the centrifugal 10min of 13,000rpm carefully outwells supernatant liquor;
5. add 500 μ l F liquid in above-mentioned centrifuge tube, the centrifugal 5min of 13,000rpm carefully abandons supernatant, comes again;
6. dry rear adding 30 μ l G liquid dissolving in room temperature; Be template DNA solution to be measured.
7. get the PCR pipe of 2+ sample size, mark good (carrying out mark according to the sample of measuring), the H liquid that adds respectively 19 μ l, and the liquid (template DNA solution to be measured), I liquid and the J liquid 1 μ l that got respectively for the 6th step join in the PCR pipe of correspondence markings number, centrifugal 30 seconds of the rear 5000rpm that is mixed places on the PCR instrument.
Increase by following condition: 95 ℃ of sex change 5min → 95 ℃, 30sec; 58 ℃, 30sec; 72 ℃, 30sec (30 circulations) → 72 ℃, 10min → 4 ℃ preservation.
8. after reaction finished, the tetrabromophenol sulfonphthalein that adds 4 μ l in each PCR was mixed, and the sepharose of preparation 1% is sequentially added into respectively the PCR reaction solution of 15 μ l in the sepharose well, and an other hole adds the DNAmarker II of 8 μ l, 120V electrophoresis 20min.
9. after electrophoresis finishes, under ultraviolet lamp, observe the contrast positive and negative control, if sample has band at the molecular weight place of primer S1:681bp and primer S2:568bp, show that this corresponding giant salamander group infects irido virus, otherwise do not infect, the results are shown in accompanying drawing 1, through the detection display of sample that 10 giant salamanders are casted off a skin, 6,8, No. 9 giant salamander group corresponding to sample do not have irido virus to infect, and 3,4,5,7,10,11, No. 12 giant salamander group corresponding to sample infected irido virus; Set up positive control (1 swimming lane) and negative control (2 swimming lane) to illustrate that method of the present invention and detection system are reliable.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (1)
1. a utilization is casted off a skin and is detected the test kit of giant salamander group irido virus band poison situation, it is characterized in that, comprises DNA extraction agent combination and pcr amplification agent combination,
Described DNA extraction agent combination comprises:
A liquid, the sample lysate of casting off a skin, 1 pipe contains PBS; 1%Triton X-100;
B liquid, 1 pipe, 200 μ g/mL Proteinase Ks;
C liquid, 1 pipe, the template extract contains phenol/chloroform/primary isoamyl alcohol, and ratio is 25: 24: 1;
D liquid, 1 pipe, 3mol/L sodium acetate;
E liquid, 1 pipe contains dehydrated alcohol;
F liquid, 1 pipe contains 75% ethanol;
G liquid, 1 pipe contains the sterilization ultrapure water;
Described pcr amplification agent combination comprises:
H liquid, the S1 pipe contains pcr amplification reaction liquid, comprises deionized water, and the dNTP substrate contains Mg
2+1 * damping fluid; The Taq enzyme; Primer S15 '-CCCCTCCCATTCTTCTTCTCC-3 ' and 5 '-GGCGTTGGTCAGTCTACCGTAAT-3 ';
The S2 pipe contains pcr amplification reaction liquid, comprises deionized water, and the dNTP substrate contains Mg
2+1 * damping fluid; The Taq enzyme; Primer S25 '-GGGCTAATGTATTGAAGACGC-3 ' and 5 '-TTGTAAACTTGGAGTGGAGGG-3;
I liquid, 1 pipe, positive control sample;
J liquid, 1 pipe, negative control product;
K liquid, 1 pipe, DNA markerII.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010550620 CN102031307B (en) | 2010-11-19 | 2010-11-19 | Kit for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010550620 CN102031307B (en) | 2010-11-19 | 2010-11-19 | Kit for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102031307A CN102031307A (en) | 2011-04-27 |
| CN102031307B true CN102031307B (en) | 2013-01-30 |
Family
ID=43884759
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010550620 Expired - Fee Related CN102031307B (en) | 2010-11-19 | 2010-11-19 | Kit for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102031307B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN105494257B (en) * | 2015-12-29 | 2018-05-22 | 贵州大学 | The method for promoting the method for giant salamander husking and extracting giant salamander genome |
| CN111647664A (en) * | 2020-06-19 | 2020-09-11 | 河北大学 | Method for non-invasive identification of insect genotype |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1448517A (en) * | 2003-05-06 | 2003-10-15 | 中山大学 | Prawn white spot complex virogene diagnostic kit and detecting method thereof |
| CN101845516A (en) * | 2009-12-04 | 2010-09-29 | 山东出入境检验检疫局检验检疫技术中心 | Real-time quantitative PCR detection method for red-sea bream iridovirus |
| CN102146483A (en) * | 2010-02-10 | 2011-08-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Loop-mediated isothermal amplification (LAMP) detection method and kit of soft-shelled turtle iridovirus (STIV) |
-
2010
- 2010-11-19 CN CN 201010550620 patent/CN102031307B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1448517A (en) * | 2003-05-06 | 2003-10-15 | 中山大学 | Prawn white spot complex virogene diagnostic kit and detecting method thereof |
| CN101845516A (en) * | 2009-12-04 | 2010-09-29 | 山东出入境检验检疫局检验检疫技术中心 | Real-time quantitative PCR detection method for red-sea bream iridovirus |
| CN102146483A (en) * | 2010-02-10 | 2011-08-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Loop-mediated isothermal amplification (LAMP) detection method and kit of soft-shelled turtle iridovirus (STIV) |
Non-Patent Citations (6)
| Title |
|---|
| 佚名.科研人员首次分离到大鲵出血病原——大鲵虹彩病毒.《水产养殖》.2011,(第3期),54. |
| 患病中国大鲵中分离到一株虹彩病毒及其特性的研究;江育林 等;《病毒学报》;20110531;第27卷(第3期);274-282 * |
| 江育林 等.患病中国大鲵中分离到一株虹彩病毒及其特性的研究.《病毒学报》.2011,第27卷(第3期),274-282. |
| 科研人员首次分离到大鲵出血病原——大鲵虹彩病毒;佚名;《水产养殖》;20110331(第3期);54 * |
| 耿毅 等.蛙病毒感染致养殖大鲵大规模死亡的电镜观察及PCR 检测.《中国兽医科学》.2010,第40卷(第8期),817-821. |
| 蛙病毒感染致养殖大鲵大规模死亡的电镜观察及PCR 检测;耿毅 等;《中国兽医科学》;20100831;第40卷(第8期);817-821 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102031307A (en) | 2011-04-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Viarouge et al. | Identification of bluetongue virus and epizootic hemorrhagic disease virus serotypes in French Guiana in 2011 and 2012 | |
| Ito et al. | Growth of cyprinid herpesvirus 2 (CyHV-2) in cell culture and experimental infection of goldfish Carassius auratus | |
| Sharawi et al. | The utility of polymerase chain reaction for diagnosis of lumpy skin disease in cattle and water buffaloes in Egypt | |
| Liang et al. | Understanding the research advances on lumpy skin disease: A comprehensive literature review of experimental evidence | |
| Crane et al. | First isolation of an aquatic birnavirus from farmed and wild fish species in Australia | |
| Esmailzadeh Hosseini et al. | The first report of a phytoplasma associated with pot marigold phyllody in Iran | |
| CN109609688A (en) | Goose astrovirus, goose's paramyxovirus, goose parvovirus multiple PCR detection primer pair and detection method and application | |
| CN103667494B (en) | A kind of detection method of sweet potato black rot pathogen | |
| Bidokhti et al. | Tracing the transmission of bovine coronavirus infections in cattle herds based on S gene diversity | |
| CN102031307B (en) | Kit for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis | |
| CN101775443B (en) | LAMP kit for detecting PRV and preparation method thereof | |
| Armson et al. | Detection of foot-and-mouth disease virus in milk samples by real-time reverse transcription polymerase chain reaction: optimisation and evaluation of a high-throughput screening method with potential for disease surveillance | |
| CN103695561B (en) | RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) nucleic acid test strip kit for detecting Japanese B encephalitis virus and application of kit | |
| CN103276104B (en) | RT-LAMP nucleic acid test strip kit for detection of porcine reproductive and respiratory syndrome virus, and applications | |
| CN103525951A (en) | LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof | |
| CN101878756B (en) | Method for storing monochamus alternatus hope sample and method for quickly detecting bursaphelenchus xylophilus therein | |
| CN102465175A (en) | Quick Clavibacter michiganensis subsp. michiganensis IC-PCR detection kit, its preparation method and its application method | |
| Rolesu et al. | Geographic information systems: a useful tool to approach African swine fever surveillance management of wild pig populations | |
| CN108950084A (en) | It is a kind of for detecting the primer sets and kit of Porcine epidemic diarrhea virus and pig astrovirus | |
| Al-Bayati | Assessment of Contagious Ecthyma Virus in Camels of Wasit Province, Iraq | |
| CN101792799A (en) | PCR quick detection method for bee European foul brood | |
| CN105063040B (en) | Phytophthora infestans germ PCR detection primers, kit and detection method | |
| Ganguly et al. | Studies on seroprevalence of infectious bovine rhinotracheitis in cattle population of West Bengal | |
| CN101319253A (en) | Method for detecting high-pathogenicity blue ear disease | |
| CN105039331A (en) | Peronophythora litchi LAMP primer as well as rapid detection method and application thereof |
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 | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130130 Termination date: 20131119 |