CN105176979A - Method for detecting infectious pancreatic necrosis virus - Google Patents
Method for detecting infectious pancreatic necrosis virus Download PDFInfo
- Publication number
- CN105176979A CN105176979A CN201510548476.3A CN201510548476A CN105176979A CN 105176979 A CN105176979 A CN 105176979A CN 201510548476 A CN201510548476 A CN 201510548476A CN 105176979 A CN105176979 A CN 105176979A
- Authority
- CN
- China
- Prior art keywords
- necrosis virus
- primer
- primer pair
- infectious
- seqidno
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000710921 Infectious pancreatic necrosis virus Species 0.000 title abstract description 11
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 6
- 241000700605 Viruses Species 0.000 claims description 24
- 208000015181 infectious disease Diseases 0.000 claims description 23
- 230000002458 infectious effect Effects 0.000 claims description 22
- 230000017074 necrotic cell death Effects 0.000 claims description 20
- 210000000496 pancreas Anatomy 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 230000003321 amplification Effects 0.000 claims description 9
- 230000004087 circulation Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 8
- 239000002299 complementary DNA Substances 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000010839 reverse transcription Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 4
- 239000013641 positive control Substances 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 230000004044 response Effects 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000011896 sensitive detection Methods 0.000 abstract description 2
- 206010028851 Necrosis Diseases 0.000 description 15
- 241000251468 Actinopterygii Species 0.000 description 10
- 235000019688 fish Nutrition 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 241000158512 Redspotted grouper nervous necrosis virus Species 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 241000711804 Infectious hematopoietic necrosis virus Species 0.000 description 3
- 241000546112 Infectious salmon anemia virus Species 0.000 description 3
- 241000711825 Viral hemorrhagic septicemia virus Species 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 241000966621 Megalocytivirus Species 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 208000007502 anemia Diseases 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 208000009663 Acute Necrotizing Pancreatitis Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 206010010947 Coordination abnormal Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000701372 Iridovirus Species 0.000 description 1
- 241001505329 Lymphocystis disease virus 1 Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 206010058096 Pancreatic necrosis Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 241000433805 Turbot reddish body iridovirus Species 0.000 description 1
- 208000001449 Viral Hemorrhagic Septicemia Diseases 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 208000013435 necrotic lesion Diseases 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 206010037628 pylorospasm Diseases 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 208000018655 severe necrosis Diseases 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting an infectious pancreatic necrosis virus. The sequences of upstream and downstream primers adopted by the method are respectively shown as SEQ ID NO:1 and SEQ ID NO:2. According to the invention, a large number of primers are designed according to the genomic sequences of the infectious pancreatic necrosis virus, and the primers capable of rapidly and effectively detecting are screened. The primers used by the method disclosed by the invention have the characteristics of high specificity, high sensitivity, high practicability and high convenience, and can carry out highly-sensitive detection on the infectious pancreatic necrosis virus.
Description
Technical field
The invention belongs to aquatic products disease technical field of microbial detection, be specifically related to a kind of method detecting infectious pancreas necrosis virus.
Background technology
Along with socioeconomic fast development, culture fishery also presents the unprecedented raising of cultivation scale and cultural technique, and especially in intensive culture and industrialized culture, development is rapid, and this brings nutrition and health to the life of the people.But in the breeding process of marine fish, breaking out of virus disease often carrys out huge harm to cultivation industrial belt.Infectious pancreatic necrosis is the fish transmissible disease caused by infectious pancreatic necrosis virus (infectiouspancreaticnecrosisvirus, IPNV), and the son of main harm salmon fishes, juvenile fish, mortality ratio reaches more than 90%.One of feature of morbidity fish is seed sudden death, and sick fish is first slowly travelling, asynergia, or following current levitating, and normal to do vertical revolution travelling, just sinks under water soon and dead.Sick fish body colour blackout, ophthalmoptosis, belly expands, belly and fin ray portion congested, the gill is incarnadine, and anus place often holds in the palm a long and thicker white mucus ight soil.Dissect disease fish and check there is ascites as seen, sick fish pancreas is congested, and pylorospasm is hemorrhage, histocyte severe necrosis; The pale anaemia of liver,spleen,kidney, also has necrotic lesion, gastrorrhagia, without food in enteron aisle, and has milky or faint yellow mucus.Therefore, detect whether aquaculture water, apparatus and feed are subject to infectious pancreatic necrosis virus in time just extremely important.But detection method mainly histopathology technology, electron microscopy, cell culture technology, immunoassay technology and the molecular detection technology etc. of IPNV at present.These detection methods have respective advantage in accuracy, susceptibility and cost etc., but they are subject to the restriction needing professional and complex instrument in varying degrees.
Summary of the invention
The object of this invention is to provide a kind of method detecting infectious pancreas necrosis virus, thus make up the deficiencies in the prior art.
First the present invention provides a kind of primer detecting infectious pancreas necrosis virus, and the sequence of its upstream and downstream primer is respectively SEQIDNO:1 and SEQIDNO:2;
As preferably, the sequence of its downstream primer is SEQIDNO:3.
The present invention also provides a kind of test kit detecting infectious pancreas necrosis virus, and this test kit includes above-mentioned primer pair.
The present invention also provides a kind of method detecting infectious pancreas necrosis virus, is whether to have infected the method for infectious pancreas necrosis virus for detecting in water body or feed, and the method comprises following step:
1) nucleic acid of detected sample is extracted as the template detected;
2) RNA of extraction is carried out reverse transcription and prepare cDNA;
3) carry out the pcr amplification detecting sample, reaction system 50 μ L, includes the Taq enzyme of 0.05U/ μ L, Mg
2+concentration is the primer of the dNTPs of the 10 × PCRBuffer of 2mmol/L, 0.25mmol/L, 0.36 μm of ol/L; Step 2) cDNA for preparing is as amplification template;
The response procedures of pcr amplification is as follows: 94 DEG C of 5min; 94 DEG C of 15s, 56 DEG C of 15s, 72 DEG C 15s, 25-40 circulation; 4 DEG C of termination reactions;
4) product carries out electrophoresis detection, has determined whether target fragment.
Wherein step 3) in carry out simultaneously positive control and negative control amplification; Positive control is the plasmid carrying infectious pancreas necrosis virus nucleic acid fragment, and negative control is deionized water.
The present invention devises a large amount of primer according to infectious pancreas necrosis virus genome sequence, therefrom filters out the primer that can effectively detect fast.Primer used in the present invention has higher specificity, sensitivity, practicality and convenience, effectively can carry out high-sensitive detection to infectious pancreas necrosis virus.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1: the design of primer
US National biology information technology center (NCBI) is logged in by Internet, the infectious pancreas necrosis virus gene nucleotide series that query and search has been announced, do not comprise not clear base content sequence, to the diseased plant of homology nearer same time with place, edit with the N gene nucleotide series of Editseq and the MegAlign software in DNAStar7.1 software package to selected sample, comparison one by one, with ClustalWMethod (software) default parameters, tetraploid rice is carried out to selected N nucleotide sequence, find the specific nucleotide sequence (target detection sequence) characterizing this virus.
Pcr amplification design of primers is undertaken by AssayDesignSW software, one group of higher primer of score is used to test, according to the homology analysis result of DNASTAR, choose sequence area comparatively conservative in sample gene, design of amplification primers, so that amplify the single band of specificity, for follow-up order-checking lays the foundation.And adopt primer-design software Primer_Premier_5.0 to analyze to the primer of design, comprise
△the design of primers indexs such as G value, primer length and GC content, select the conduct primer pair used in the present invention that mark is the highest, wherein the sequence of upstream primer is 5 '-CAAACAAAGCAACCGCAAC-3 ' (SEQIDNO:1), the sequence of downstream primer is 5 '-GTCCCATTCAGGGCATAGAG-3 ' (SEQIDNO:2), the annealing temperature of primer is preferably 54 DEG C, and expanding fragment length is 352bp.
Embodiment 2: the specific detection of primer
In order to verify the specificity of primer of the present invention, choose and carry lymphocystis disease virus LCDV, Megalocytivirus belongs to irido virus (Megalocytivirus, Mega), red-spotted grouper nervous necrosis virus (red-spottedgroupernervousnecrosisvirus, RGNNV), infectious hematopoietic necrosis's poison (infectioushaematopoieticnecrosisvirus, IHNV), viral hemorrhagic septicemia, VHS virus (viralhemorrhagicsepticemiavirus, and Infectious Salmonia Anemia virus (infectioussalmonanaemiavirus VHSV), ISAV) plasmid of nucleic acid fragment is as template.
The step detected is as follows:
(1) extraction of sample tissue nucleic acid.Sample thief about 0.02 ~ 1g is placed in 1.5ml plastic, with disposable grinding rod, sample is milled to pulpous state fast, then DNA extraction kit (Tian Gen biochemical technology company limited is used, Beijing) and TRizol (precious biotinylated biomolecule Engineering Co., Ltd, Dalian) extract DNA and RNA respectively.Concrete operation step carries out according to the specification sheets of test kit or reagent.If the viral sample plasmid bought, then omit this step
(2) cDNA is prepared in reverse transcription.When detecting the RNA viruses such as RGNNV, IHNV, IPNV, VHSV, ISAV, need to carry out reverse transcription to RNA template, use the single stage method gDNA of Beijing Quan Shijin biotech firm removal and cDNA synthetic agent box to carry out.Concrete grammar is: in the Eppendorf tube of 200 μ l, adds 5 ~ 500ngRNA sample, 1 μ l random primer (0.1 μ g/ μ l), 10 μ l reverse transcription buffer (2 × TSReactionMix), 1 μ l ThermoScript II (
rT/RIEnzymeMix), 1 μ l genomic dna degrading enzyme (gDNARemover), DEPC process water complements to 20 μ l.Above-mentioned reaction system is placed in 25 DEG C of 10min, then 42 DEG C of 30min, then 85 DEG C of 5min, last 4 DEG C save backup.Detect the DNA virus such as IPNV, Mega, TRBIV and do not need reverse transcription, directly use the DNA masterplate extracted.
(3) pcr amplification
In the PCR reaction tubes of single 200 μ l, containing 2.5U/ μ LTaqDNA polysaccharase 0.6 μ L, 10 × PCRBuffer (Mg
2+plus) 5 μ L, 2.5mmol/LdNTPs5 μ L, 2 μm of ol/LPrimer5 μ L, the DNA profiling of extraction and the cDNA masterplate of preparation each 1 μ L, ddH
2o is supplemented to 50 μ L.Amplified reaction program: 94 DEG C of 5min; 94 DEG C of 15s, 54 DEG C of 15s, 72 DEG C of 15s, 15 circulations; 94 DEG C of 15s, 70 DEG C of 15s, 6 circulations; 72 DEG C of 3min; 4 DEG C of preservations.
Product is carried out agarose gel electrophoresis, and adopt 2.0% sepharose to be separated PCR primer, 4SRed nucleic acid dye is added in the gel of thawing to specifications.To take pictures under gel imaging system after 90V constant voltage electrophoresis 35min observation.
Electrophoresis result proves to only have the swimming lane of the sample of infectious pancreas necrosis virus to have amplified production, thus proves that primer of the present invention has good specific amplification.
Embodiment 3: the mensuration of primer detection sensitivity
Get 1 μ L10 times gradient dilution (10
4~ 10
1, 5copies/ μ L) containing the plasmid of infectious pancreas necrosis virus nucleic acid as template, carry out the mensuration of sensitivity by the method for embodiment 2.Result shows, primer of the present invention just can can detect the plasmid of 10 copies 27 amplification cycles; But for 5 copy, need 38 circulations to show amplified band.
In order to improve sensitivity during detection, downstream primer being modified optimization, obtains new primer sequence 5 '-GTCCGATTCAGTGCATAGAG-3 ' (SEQIDNO:3); Be analyzed according to above-mentioned specificity and susceptibility detecting step, found that: compared to the primer amplification 10ng template before not optimizing, amplified band is more obviously clear under same cycle index condition.And, under similarity condition, primer before optimization 35 circulation times can not from 5 copy plasmid template sample amplify can be visual in gel electrophoresis product, and the primer after optimizing can go out at 32 cyclic amplifications the plasmid template sample that minimum concentration is 5 copies, and there is no the appearance of primer dimer.Illustrate that the primer after optimizing can significantly improve sensitivity, and specificity does not receive any impact.
The detection of embodiment 4 aquaculture water
The water body adopting primer pair of the present invention to there occurs infectious pancreas necrosis virus detects, and has carried out synchronous detection to the sick fish sample of 30 tails collected simultaneously.Result shows, and use the upstream primer that sequence is SEQIDNO:1, and the downstream primer of SEQIDNO:2 increases, and whole sick fish sample just detects object band at 25 circulation times; And water body just can will detect 39 circulations.Adopt sequence to be that the downstream primer of SEQIDNO:3 and the upstream primer of SEQIDNO:1 increase, obtain positive band 35 circulations with regard to obtaining to increase; Thus avoid the generation of false negative result.
Claims (7)
1. detect a primer pair for infectious pancreas necrosis virus, it is characterized in that, the sequence of the upstream and downstream primer of described primer pair is respectively SEQIDNO:1 and SEQIDNO:2.
2. primer pair as claimed in claim 1, it is characterized in that, the sequence of described downstream primer is SEQIDNO:3.
3. detect a test kit for infectious pancreas necrosis virus, it is characterized in that, described test kit includes the primer pair described in claim 1 or 2.
4. detect a method for infectious pancreas necrosis virus, be whether infected the method for infectious pancreas necrosis virus for detecting in water body or feed, the method comprises following step:
1) nucleic acid of detected sample is extracted as the template detected;
2) RNA of extraction is carried out reverse transcription and prepare cDNA;
3) carry out the pcr amplification detecting sample, reaction system 50 μ L, includes the Taq enzyme of 0.05U/ μ L, Mg
2+concentration is the dNTPs of the 10 × PCRBuffer of 2mmol/L, 0.25mmol/L, the primer pair described in the claim 1 or 2 of 0.36 μm of ol/L; Step 2) cDNA for preparing is as amplification template;
The response procedures of pcr amplification is as follows: 94 DEG C of 5min; 94 DEG C of 15s, 56 DEG C of 15s, 72 DEG C 15s, 25-35 circulation; 4 DEG C of termination reactions;
4) product carries out electrophoresis detection, has determined whether target fragment.
5. method as claimed in claim 4, is characterized in that, described step 3) in carry out positive control and negative control amplification simultaneously.
6. method as claimed in claim 5, it is characterized in that, described positive control is the plasmid carrying infectious pancreas necrosis virus nucleic acid fragment.
7. method as claimed in claim 5, it is characterized in that, described negative control is deionized water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510548476.3A CN105176979B (en) | 2015-08-31 | 2015-08-31 | A method of detection infectious pancreas necrosis virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510548476.3A CN105176979B (en) | 2015-08-31 | 2015-08-31 | A method of detection infectious pancreas necrosis virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105176979A true CN105176979A (en) | 2015-12-23 |
| CN105176979B CN105176979B (en) | 2018-10-26 |
Family
ID=54899409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510548476.3A Active CN105176979B (en) | 2015-08-31 | 2015-08-31 | A method of detection infectious pancreas necrosis virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105176979B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112176111A (en) * | 2020-11-05 | 2021-01-05 | 北京市水产技术推广站 | Primer, probe, kit and detection method for detecting infectious pancreatic necrosis virus |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687447A (en) * | 2005-04-12 | 2005-10-26 | 中华人民共和国连云港出入境检验检疫局 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
| CN101906486A (en) * | 2010-08-03 | 2010-12-08 | 中国水产科学研究院黄海水产研究所 | Gene chip for detecting various fish pathogens and detecting method thereof |
| CN103937912A (en) * | 2014-05-13 | 2014-07-23 | 吴斌 | LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition |
-
2015
- 2015-08-31 CN CN201510548476.3A patent/CN105176979B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687447A (en) * | 2005-04-12 | 2005-10-26 | 中华人民共和国连云港出入境检验检疫局 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
| CN101906486A (en) * | 2010-08-03 | 2010-12-08 | 中国水产科学研究院黄海水产研究所 | Gene chip for detecting various fish pathogens and detecting method thereof |
| CN103937912A (en) * | 2014-05-13 | 2014-07-23 | 吴斌 | LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting infectious pancreas necrosis virus and application of LAMP primer composition |
Non-Patent Citations (3)
| Title |
|---|
| GENBANK: "AF160258.1", 《GENBANK》 * |
| 吴斌 等: "传染性胰脏坏死病毒逆转录聚合酶链式反应法（RT-PCR）的研究", 《中国动物检疫》 * |
| 徐晔 等: "实时荧光定量RT-PCR检测鱼类传染性胰脏坏死病病毒方法的建立", 《安徽农业科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112176111A (en) * | 2020-11-05 | 2021-01-05 | 北京市水产技术推广站 | Primer, probe, kit and detection method for detecting infectious pancreatic necrosis virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105176979B (en) | 2018-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Thu et al. | DNA barcoding of coastal ray-finned fishes in Vietnam | |
| Christiansen et al. | A low-pathogenic variant of infectious salmon anemia virus (ISAV-HPR0) is highly prevalent and causes a non-clinical transient infection in farmed Atlantic salmon (Salmo salar L.) in the Faroe Islands | |
| Kuo et al. | Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture | |
| Mohr et al. | New yellow head virus genotype (YHV7) in giant tiger shrimp Penaeus monodon indigenous to northern Australia | |
| Huang et al. | Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in C hina | |
| Vij et al. | Barcoding of Asian seabass across its geographic range provides evidence for its bifurcation into two distinct species | |
| CN107385111A (en) | The real-time fluorescence quantitative PCR detection primer and its kit of a kind of goose astrovirus | |
| Sakuna et al. | A novel virus (order Bunyavirales) from stressed redclaw crayfish (Cherax quadricarinatus) from farms in northern Australia | |
| Mullen et al. | Nuclear small-subunit ribosomal RNA gene-based characterization, molecular phylogeny and PCR detection of the Neoparamoeba from western Long Island Sound lobster | |
| CN114657264B (en) | Clarias fuscus sex-specific molecular marker primer and application thereof | |
| CN106282377B (en) | A kind of Transgenic salmon AquAdvantage strain specificity real-time fluorescent PCR testing primer, detection method and kit | |
| Ma et al. | Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers | |
| CN105543415A (en) | Nest type PCR detection method for different variants of ostreid herpes virus | |
| Chakrabarty et al. | Assessment of WSSV prevalence and distribution of disease-resistant shrimp among the wild population of Penaeus monodon along the west coast of India | |
| Zhang et al. | Construction of a genetic linkage map for silver carp (Hypophthalmichthys molitrix) | |
| Chi et al. | High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species | |
| Moore et al. | Impact of poxvirus lesions on saltwater crocodile (Crocodylus porosus) skins | |
| CN102586451B (en) | Method for matching and breeding jian carps | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| Jiang et al. | A microsatellite genetic linkage map of half smooth tongue sole (Cynoglossus semilaevis) | |
| CN105176979A (en) | Method for detecting infectious pancreatic necrosis virus | |
| CN104962660A (en) | Ruditapes philippinarum species real-time fluorescent PCR (polymerase chain reaction) specific detection system and application thereof | |
| CN105002303B (en) | A kind of PCR method for detecting lymphocystis disease virus | |
| CN104204231A (en) | Real-time viral detection method and kit for infectious hypodermaland haematopoietic necrosis virus | |
| CN111471774B (en) | Co-dominant long INDEL molecular marker for sex discrimination of cynoglossus semilaevis and method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180814 Address after: 518000 Guangdong Shenzhen Longhua New District big wave street Longsheng community Tenglong road gold rush e-commerce incubation base exhibition hall E commercial block 706 Applicant after: Shenzhen step Technology Transfer Center Co., Ltd. Address before: 266001 47, B 104, Fulong Road, Shinan District, Qingdao, Shandong Applicant before: Sun Cuiyan |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20180914 Address after: 362300 No. 97, Shun Mei Shun Road, Nanan, Fujian. Applicant after: Shi Shuqin Address before: 518000 Guangdong Shenzhen Longhua New District big wave street Longsheng community Tenglong road gold rush e-commerce incubation base exhibition hall E commercial block 706 Applicant before: Shenzhen step Technology Transfer Center Co., Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191213 Address after: Room 1602, B1 Floor, Lijinxiu Jiangnan (West District), east of Gouyun Road, north of Yinhe No. 2 Road, Suzhou City, Anhui Province Patentee after: Suzhou Huiteng Intellectual Property Consulting Co., Ltd. Address before: 362300 Nanan, Fujian, the United States and the United States along the River Road, No. 97 Patentee before: Shi Shuqin |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200721 Address after: Room 501, building 6, no.371, Hongye Road, Dayun Town, Jiashan County, Jiaxing City, Zhejiang Province Patentee after: Zhejiang Hengyu Biotechnology Co., Ltd Address before: Room 1602, B1 Floor, Lijinxiu Jiangnan (West District), east of Gouyun Road, north of Yinhe No. 2 Road, Suzhou City, Anhui Province Patentee before: Suzhou Teng Teng Intellectual Property Advisory Co.,Ltd. |