CN101805788B - 基于scar分子标记快速检测副溶血弧菌的pcr特异扩增引物及检测试剂盒 - Google Patents
基于scar分子标记快速检测副溶血弧菌的pcr特异扩增引物及检测试剂盒 Download PDFInfo
- Publication number
- CN101805788B CN101805788B CN2009102490644A CN200910249064A CN101805788B CN 101805788 B CN101805788 B CN 101805788B CN 2009102490644 A CN2009102490644 A CN 2009102490644A CN 200910249064 A CN200910249064 A CN 200910249064A CN 101805788 B CN101805788 B CN 101805788B
- Authority
- CN
- China
- Prior art keywords
- reagent
- primer
- vibrio parahaemolyticus
- detection
- vibrio
- 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.)
- Active
Links
- 241000607272 Vibrio parahaemolyticus Species 0.000 title claims abstract description 30
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 231100000241 scar Toxicity 0.000 title claims abstract 4
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 44
- 238000011144 upstream manufacturing Methods 0.000 claims description 14
- 230000003321 amplification Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 239000003550 marker Substances 0.000 claims description 8
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 239000012160 loading buffer Substances 0.000 claims description 3
- 241000607598 Vibrio Species 0.000 abstract description 13
- 241001465754 Metazoa Species 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 abstract 1
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 239000012154 double-distilled water Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 206010047400 Vibrio infections Diseases 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 241000694873 Paralichthyidae Species 0.000 description 4
- 208000008469 Peptic Ulcer Diseases 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 241000607618 Vibrio harveyi Species 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000607594 Vibrio alginolyticus Species 0.000 description 2
- 241000607323 Vibrio campbellii Species 0.000 description 2
- 241000607253 Vibrio mimicus Species 0.000 description 2
- 241000607265 Vibrio vulnificus Species 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- 241000607291 Vibrio fluvialis Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002969 morbid Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种基于SCAR分子标记快速检测副溶血弧菌的PCR特异扩增引物及检测试剂盒。上、下游引物的核苷酸序列为：上游引物VpaF：5′-ACTACCGAACACGGGCTGTAT-3′；下游引物VpaR：5′-GGCTGGGCATCGTAATCAT-3′。试剂盒中包括用灭菌双蒸水溶解的PCR特异扩增上游引物VpaF和下游引物VpaR的混合液，浓度为0.5-2μM。可用培养的弧菌菌落直接做PCR反应进行检测，无需提取弧菌基因组DNA，省时省力、简便、快速、准确、特异性好、灵敏度高。该试剂盒可用于海水养殖动物养殖过程中副溶血弧菌的跟踪检测及海水环境监测，具有很高的实用价值。
Description
技术领域：
本发明涉及一种检测海水养殖动物病原菌的PCR特异扩增引物及检测试剂盒，尤其是一种检测准确性及灵敏性高、时间短、成本低的基于SCAR(Sequence characterized amplified region，特征性序列扩增区域)分子标记快速检测副溶血弧菌的PCR特异扩增引物及检测试剂盒。
背景技术：
弧菌是海水养殖动物主要的条件致病菌，其中病原性弧菌能使养殖动物暴发性死亡，与病毒、其他细菌或寄生虫等一起将会对海水养殖动物造成巨大的损害，被公认为是海水养殖中最为严重的病害之一。如副溶血弧菌(Vibrioparahaemolyticus)可导致鱼、虾、贝、海参等养殖动物大规模死亡，也给误食感染了副溶血弧菌食物的人类健康带来隐患。目前，对弧菌的检测方法有生理生化鉴定技术、酶联免疫吸附试验、16S rRNA基因鉴定等，分别存在如下不足：生理生化鉴定技术耗时费力，检测的准确率低；酶联免疫吸附试验敏感性不高；16S rRNA基因鉴定只能将致病弧菌鉴定到属，而无法鉴定到种。
发明内容：
本发明是针对现有技术所存在的上述技术问题，提供一种检测准确性及灵敏性高、时间短、成本低的基于SCAR分子标记快速检测副溶血弧菌的PCR特异扩增引物及检测试剂盒。
本发明的技术解决方案是：一种基于SCAR分子标记快速检测副溶血弧菌的PCR特异扩增引物，其特征在于上、下游引物的核苷酸序列为：
上游引物VpaF：5′-ACTACCGAACACGGGCTGTAT-3′；
下游引物VpaR：5′-GGCTGGGCATCGTAATCAT-3′。
一种基于SCAR分子标记快速检测副溶血弧菌的检测试剂盒，其特征在于包含如下试剂：
试剂I：用灭菌双蒸水溶解的PCR特异扩增上游引物VpaF和下游引物VpaR的混合液，浓度为0.5-2μM，上、下游引物的核苷酸序列为：上游引物VpaF：5′-ACTACCGAACACGGGCTGTAT-3′；下游引物VpaR：5′-GGCTGGGCATCGTAATCAT-3′
试剂II：dNTPs四种脱氧核苷酸的混合液，浓度为50～500μM；
试剂III：10×PCR缓冲液：500mM KCl、pH 8.4的100mM Tris-HCl、15mM MgCl2和16mM二硫苏糖醇；
试剂IV：Taq DNA聚合酶，浓度为5U/μl；
试剂V：灭菌双蒸水；
试剂VI：作为阳性模板的副溶血弧菌的总DNA，浓度为50ng/μl；
试剂VII：含有1×上样缓冲液的DL2000DNA分子量，浓度为50ng/μl。
本发明是利用RAPD(Random amplified polymorphic DNA，随机扩增多态性DNA)技术在各种弧菌中进行分析，发现了副溶血弧菌的差异特异性DNA片段，将差异特异性DNA片段回收测序，然后再根据测得的序列转化成SCAR标记。设计并合成了用于检测副溶血弧菌的PCR特异扩增上下游引物，组装检测试剂盒，可用培养的弧菌菌落直接做PCR反应进行检测，无需提取弧菌基因组DNA，可以将弧菌鉴定到种。省时省力、简便、快速、准确、特异性好、灵敏度高。可用于海水养殖动物养殖过程中副溶血弧菌的跟踪检测及海水环境监测，具有很高的实用价值。
附图说明：
图1是用本发明实施例检测患溃疡病的牙鲆病灶的结果示意图。
具体实施方式：
本发明实施例是利用200多条RAPD引物在副溶血弧菌(Vibrioparahaemolyticus)(ATCC 17802)、创伤弧菌(Vibrio vulnificus)(ATCC 27562)、溶藻弧菌(Vibrio alginolyticus)(ATCC 17749)、河弧菌(Vibrio fluvialis)(ATCC33810)、最小弧菌(Vibrio mimicus)(ATCC 33653)、哈氏弧菌(Vibrio harveyi)(ATCC 33842)、坎氏弧菌(Vibrio campbellii)(ATCC 33864)和鲨鱼弧菌(Vibriocarchariae)(ATCC 35084)等弧菌上开发并转化为副溶血弧菌的种属特异性SCAR标记，其核苷酸序列为：
ACTACCGAACACGGGCTGTATACGTTTAGCGAAATGGATAGAAATGAAGGTGCAGAGCTCGCGTTTTTTCGCGGTAGTGAATCCGTCTCTTTGCCGATTAATGGCGATTATCCGTGTCATTTGGATATCAAAGAACCGTTGTTGGCGGTTGCTAATTATGGCTCTGGAAATGTTAGCGTATTTCAATTGGATCGCGATGGTAAACCGCTTGGTTTATTGGCGGATTTATACGTTGATGGATGCGGGCCGAATTTAGAGCGCCAAACTTCTCCTCACGCTCATCAAGTCACGTTTTTAAAGCACAGTCATCAATTGGTTGTCGTAGACTTAGGCAGCGATAGCGTCCTTATTTATGATTACGATGCCCAGCC。
本发明实施例是根据上述特异性SCAR标记，设计并合成了快速检测副溶血弧菌的PCR特异扩增引物，其特征在于上、下游引物的核苷酸序列为：
上游引物VpaF：5′-ACTACCGAACACGGGCTGTAT-3′；
下游引物VpaR：5′-GGCTGGGCATCGTAATCAT-3′。
本发明所设计的检测试剂盒含有如下试剂：
试剂I：用灭菌双蒸水溶解的PCR特异扩增上游引物VpaF(5′-ACTACCGAACACGGGCTGTAT-3′)和下游引物VpaR(5′-GGCTGGGCATCGTAATCAT-3′)的混合液，浓度为0.5-2μM；
试剂II：dNTPs四种脱氧核苷酸的混合液，浓度为50～500μM；
试剂III：10×PCR缓冲液：500mM KCl、pH 8.4的100mM Tris-HCl、15mM MgCl2和16mM二硫苏糖醇(DTT)；
试剂IV：Taq DNA聚合酶，浓度为5U/μl；
试剂V：灭菌双蒸水(ddH2O)；
试剂vI：作为阳性模板的副溶血弧菌(Vibrio parahaemolyticus)(ATCC17802)的总DNA，浓度为50ng/μl；
试剂VII：含有1×上样缓冲液的DL2000DNA分子量，浓度为50ng/μl。
用本发明实施例检测患溃疡病的牙鲆病灶致病性副溶血弧菌，按下述步骤进行：
(1)在超净工作台上用接菌环刮取溃疡病的牙鲆病灶，在2216E培养基上划线接菌，28℃培养10～12小时，当细菌菌落的直径在1～1.5mm左右时可直接用于SCAR-PCR反应；
(2)在200μl PCR反应管中放1μl试剂I、2μl试剂II、2.5μl试剂III、0.5μl试剂IV及19μl试剂V，充分混匀；
(3)用无菌牙签在2216E平板上随机挑取单菌落，再将单菌落置于上述PCR反应管中，用作细菌总DNA模版；
(4)然后将PCR管放在热循环仪上进行扩增反应，反应的条件为：94℃预变性5分钟，然后进行如下25个循环：94℃变性30秒，58℃退火25秒，72℃延伸25秒；最后72℃延伸7分钟，4℃保存；做检测样品；
(5)在200μl PCR反应管中放1μl试剂I、2μl试剂II、2.5μl试剂III、0.5μl试剂IV及19μl试剂V，充分混匀，然后将PCR管放在热循环仪上进行扩增反应，反应的条件为：94℃预变性5分钟，然后进行如下25个循环：94℃变性30秒，58℃退火25秒，72℃延伸25秒；最后72℃延伸7分钟，4℃保存；做阴性对照；
(6)在200μl PCR反应管中放1μl试剂I、2μl试剂II、2.5μl试剂III、0.5μl试剂IV、18μl试剂V及1μl试剂VI，充分混匀；然后将PCR管放在热循环仪上进行扩增反应，反应的条件为：94℃预变性5分钟，然后进行如下25个循环：94℃变性30秒，58℃退火25秒，72℃延伸25秒；最后72℃延伸7分钟，4℃保存；做阳性对照；
(7)分别取步骤(4)、(5)、(6)和试剂VII 3-5μl经1％琼脂糖凝胶电泳25-35分钟后于紫外仪上观察，若在371bp处出现明亮的DNA条带，则为副溶血弧菌阳性；若无反应条带显示，则为阴性。
实际检测结果如图1所示，图中M：DL2000DNA分子量(试剂VII)；1：副溶血弧菌阴性对照(用试剂VI作DNA模版的SCAR-PCR结果)；2：副溶血弧菌阳性对照(用试剂V的SCAR-PCR结果)；3：检测样品(用培养的细菌菌落做SCAR-PCR结果)。
结果表明所检测患溃疡病的牙鲆病灶中有副溶血弧菌。
序列表
<110>大连水产学院
<120>基于SCAR分子标记快速检测副溶血弧菌的PCR特异扩增引物及检测
试剂盒
<160>2
<170>PatentIn Version 3.1
<210>1
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>1
actaccgaac acgggctgta t 21
<210>2
<211>22
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>引物
<400>2
ggctgggcat cgtaatcat 19
Claims (2)
1.一种基于SCAR分子标记快速检测副溶血弧菌的PCR特异扩增引物，其特征在于上、下游引物的核苷酸序列为：
上游引物VpaF：5′-ACTACCGAACACGGGCTGTAT-3′；
下游引物VpaR：5′-GGCTGGGCATCGTAATCAT-3′。
2.一种基于SCAR分子标记快速检测副溶血弧菌的检测试剂盒，其特征在于包含如下试剂：
试剂I：用灭菌双蒸水溶解的PCR特异扩增上游引物VpaF和下游引物VpaR的混合液，浓度为0.5-2μM，上、下游引物的核苷酸序列为：上游引物VpaF：5′-ACTACCGAACACGGGCTGTAT-3′；下游引物VpaR：5′-GGCTGGGCATCGTAATCAT-3′；
试剂II：dNTPs四种脱氧核苷酸的混合液，浓度为50～500μM；
试剂III：10×PCR缓冲液：500mM KCl、pH8.4的100mM Tris-HCl、15mM MgCl2和16mM二硫苏糖醇；
试剂IV：Taq DNA聚合酶，浓度为5U/μl；
试剂V：灭菌双蒸水；
试剂VI：作为阳性模板的副溶血弧菌的总DNA，浓度为50ng/μl；
试剂VII：含有1×上样缓冲液的DL2000DNA分子量标准，浓度为50ng/μl。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102490644A CN101805788B (zh) | 2009-12-31 | 2009-12-31 | 基于scar分子标记快速检测副溶血弧菌的pcr特异扩增引物及检测试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102490644A CN101805788B (zh) | 2009-12-31 | 2009-12-31 | 基于scar分子标记快速检测副溶血弧菌的pcr特异扩增引物及检测试剂盒 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101805788A CN101805788A (zh) | 2010-08-18 |
| CN101805788B true CN101805788B (zh) | 2013-10-16 |
Family
ID=42607741
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102490644A Active CN101805788B (zh) | 2009-12-31 | 2009-12-31 | 基于scar分子标记快速检测副溶血弧菌的pcr特异扩增引物及检测试剂盒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101805788B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045757A (zh) * | 2012-05-17 | 2013-04-17 | 上海七海复泰生物科技有限公司 | 一种免抽提虹彩病毒pcr检测方法 |
| CN111154901B (zh) * | 2020-01-19 | 2022-05-20 | 广东省微生物研究所（广东省微生物分析检测中心） | 副溶血性弧菌特异性新分子靶标及其快速检测方法 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100540681C (zh) * | 2007-06-05 | 2009-09-16 | 宁波大学 | 一种海洋弧菌的多重pcr反应试剂盒及其检测方法 |
| CN101113473A (zh) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | 运用复合荧光pcr技术检测食源性致病弧菌的方法 |
| CN101109019A (zh) * | 2007-06-28 | 2008-01-23 | 上海交通大学 | 添有扩增内标的副溶血弧菌pcr检测方法 |
| CN101285090B (zh) * | 2008-05-21 | 2010-12-01 | 厦门市疾病预防控制中心 | 食源性致病菌的检测方法 |
-
2009
- 2009-12-31 CN CN2009102490644A patent/CN101805788B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN101805788A (zh) | 2010-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Khan et al. | Development of a rapid quantitative PCR assay for direct detection and quantification of culturable and non-culturable Escherichia coli from agriculture watersheds | |
| Shabarinath et al. | Detection and characterization of Salmonella associated with tropical seafood | |
| CN102154497B (zh) | 食品中霍乱弧菌、副溶血性弧菌和沙门氏菌的m-PCR引物和探针及检测方法 | |
| CN102367475B (zh) | 不同血清型霍乱弧菌的m-PCR检测试剂盒及检测方法 | |
| Churruca et al. | Detection of Campylobacter jejuni and Campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons | |
| US20090226895A1 (en) | Method of detecting vibrio parahaemolyticus via real-time PCR-hybridization | |
| CN102260744A (zh) | 一种特异性指示禽粪便污染的基因检测方法 | |
| CN102827925A (zh) | 一种TaqMan探针荧光RT-PCR检测创伤弧菌的非诊断性方法 | |
| Mougin et al. | Development of a mreB-targeted real-time PCR method for the quantitative detection of Vibrio harveyi in seawater and biofilm from aquaculture systems | |
| US20170088882A1 (en) | Carrier for detecting foodborne-illness-causing bacteria, kit for detecting foodborne-illness-causing bacteria, method for detecting foodborne-illness-causing bacteria, and pcr reaction solution for foodborne-illness-causing bacteria | |
| Zhou et al. | Rapid visual detection of Aeromonas salmonicida by loop‐mediated isothermal amplification with hydroxynaphthol blue dye | |
| CN102337342A (zh) | 用于诊断和鉴别牛巴贝斯虫种类的试剂盒及制备方法 | |
| Bölske et al. | Diagnosis of paratuberculosis by PCR. | |
| CN101805788B (zh) | 基于scar分子标记快速检测副溶血弧菌的pcr特异扩增引物及检测试剂盒 | |
| CN101875967B (zh) | 一种食源性致病菌的快速鉴定方法 | |
| Hossain et al. | Detection of Vibrio cholerae and Vibrio vulnificus by duplex PCR specific to the groEL gene | |
| CN111154901A (zh) | 副溶血性弧菌特异性新分子靶标及其快速检测方法 | |
| CN103436623A (zh) | 一种食品中沙门氏菌活菌的快速检测试剂盒及使用方法 | |
| CN103333946B (zh) | 创伤弧菌和哈氏弧菌快速检测方法 | |
| CN101942507B (zh) | 环介导恒温扩增技术快速检测链状亚历山大藻的方法 | |
| CN101928769B (zh) | 基于scar分子标记快速检测溶藻弧菌的pcr特异扩增引物及检测试剂盒 | |
| CN102732599A (zh) | 双重聚合酶链反应方法检测海水样品中的创伤弧菌和副溶血弧菌 | |
| Mackie et al. | Experimental parameters affecting quantitative PCR of Artemia franciscana: a model for a marine zooplanktonic target in natural plankton samples | |
| CN103255202A (zh) | 霍乱弧菌、副溶血弧菌和创伤弧菌的三重pcr检测方法 | |
| CN1944665B (zh) | 用于检测迟缓爱德华氏菌的引物序列及检测试剂盒 |
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 | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: Heishijiao street Shahekou Dalian District 116000 Liaoning province No. 52 Patentee after: DALIAN OCEAN University Country or region after: China Address before: Heishijiao street Shahekou Dalian District 116000 Liaoning province No. 52 Patentee before: Dalian Fisheries University Country or region before: China |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240702 Address after: 118300 Xiaotan Village Group South, Xinglongshan Village, Yiquan Town, Donggang City, Dandong City, Liaoning Province Patentee after: Sangmo Marine Ranch Center, Yiquan Town, Donggang City Country or region after: China Address before: Heishijiao street Shahekou Dalian District 116000 Liaoning province No. 52 Patentee before: DALIAN OCEAN University Country or region before: China |