CN108432675A - 一种中华绒螯蟹活体无损取样方法及应用 - Google Patents
一种中华绒螯蟹活体无损取样方法及应用 Download PDFInfo
- Publication number
- CN108432675A CN108432675A CN201810184026.4A CN201810184026A CN108432675A CN 108432675 A CN108432675 A CN 108432675A CN 201810184026 A CN201810184026 A CN 201810184026A CN 108432675 A CN108432675 A CN 108432675A
- Authority
- CN
- China
- Prior art keywords
- eriocheir sinensis
- sampling
- spf
- live body
- body sampling
- 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
- 241000371997 Eriocheir sinensis Species 0.000 title claims abstract description 52
- 238000005070 sampling Methods 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 31
- 235000013601 eggs Nutrition 0.000 claims abstract description 13
- 230000034004 oogenesis Effects 0.000 claims abstract description 13
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 11
- 238000002955 isolation Methods 0.000 claims abstract description 5
- 241000252069 Megalops Species 0.000 claims abstract description 3
- 241000238557 Decapoda Species 0.000 claims description 9
- 241000040710 Chela Species 0.000 claims description 7
- 102000002322 Egg Proteins Human genes 0.000 claims description 4
- 108010000912 Egg Proteins Proteins 0.000 claims description 4
- 210000001015 abdomen Anatomy 0.000 claims description 4
- 210000004681 ovum Anatomy 0.000 claims description 4
- 241000371986 Eriocheir Species 0.000 claims description 3
- 238000007397 LAMP assay Methods 0.000 claims description 2
- 241000058338 Macrobrachium nipponense Species 0.000 claims description 2
- 239000004677 Nylon Substances 0.000 claims description 2
- 230000012447 hatching Effects 0.000 claims description 2
- 230000001418 larval effect Effects 0.000 claims description 2
- 229920001778 nylon Polymers 0.000 claims description 2
- 230000034303 cell budding Effects 0.000 claims 1
- 230000000366 juvenile effect Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 13
- 230000001717 pathogenic effect Effects 0.000 abstract description 12
- 244000052769 pathogen Species 0.000 abstract description 11
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 2
- 241000318927 Shrimp white spot syndrome virus Species 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 235000015170 shellfish Nutrition 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000696962 White spot syndrome virus Species 0.000 description 2
- 238000002266 amputation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 210000000514 hepatopancreas Anatomy 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001327110 Macrobrachium rosenbergii Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000713112 Orthobunyavirus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001396014 Priacanthus arenatus Species 0.000 description 1
- 241000702263 Reovirus sp. Species 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/59—Culture of aquatic animals of shellfish of crustaceans, e.g. lobsters or shrimps
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/90—Sorting, grading, counting or marking live aquatic animals, e.g. sex determination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Animal Husbandry (AREA)
- Marine Sciences & Fisheries (AREA)
- Biodiversity & Conservation Biology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种中华绒螯蟹活体无损取样方法及应用，通过对中华绒螯蟹抱卵亲本的受精卵取样，提取其DNA及RNA进行相关病原的检测，对不含检测目标病原的亲本进行隔离培育，人工繁殖获得中华绒螯蟹SPF大眼幼体。该方法具有以下优点：取样不损伤母体，保证亲蟹的正常抱卵孵幼，获得的中华绒螯蟹大眼幼体不带特定病原。本发明解决了中华绒螯蟹SPF育苗中无损活体取样的难题，为中华绒螯蟹SPF育苗提供了一条简便可行的途径，可望由此培育SPF健康苗种，提高中华绒螯蟹养殖经济效益。
Description
技术领域
本发明涉及经济甲壳类的SPF育苗领域及其它需要检测特定病原的应用领域，具体的说是对有抱卵习性的虾蟹进行无损取样方法及SPF育苗应用。
背景技术
中华绒螯蟹（Eriocheir sinensis），是我国重要的经济水产动物，又称为河蟹，广泛分布于我国大江南北各地河流湖泊。中华绒螯蟹已知的病毒包括：呼肠孤病毒（EsRV816，905及2012）、布尼亚病毒（EsBV）、及白斑综合症病毒（WSSV）等几类。这些病毒都具有垂直传播的特性。已知的中华绒螯蟹病毒在一定条件下可以使中华绒螯蟹致病，甚至致死。
由于白斑综合症病毒（WSSV）的流行，无特定病原（SPF，Specific pathogen free）育苗技术在对虾中首先得到推广应用。SPF育苗要求亲本不带特定病原，必须首先对亲本进行特定病原检测，对经济甲壳类繁育亲本进行活体取样用于分子检测时，由于甲壳类体表覆盖一层硬壳，无法进行体表取样，所以通常采用断肢取肌肉的方法，此法造成取样目标个体的伤残，活力下降，甚至部分个体感染死亡，因此，该法不适用于需要长时间抱卵的部分虾蟹种类的SPF育苗的病原检测。而如果要检测其他组织，如肝胰腺、精卵巢等需要杀死检测亲本，更不可行。以中华绒螯蟹为例，通常每年的12月交配产卵，抱卵蟹过冬，至第二年4月进行挂篮孵幼，中间经历近半年，抱卵蟹的活力对成活率非常重要，有伤残的抱卵蟹很难坚持到挂篮排幼时期。寻找有效的活体无损检测方法对中华绒螯蟹的SPF育苗非常必要。
发明内容
本发明的目的是解决具有抱卵特性的中华绒螯蟹等经济甲壳类SPF育苗中无损活体取样进行目标病原检测的难题，同时提供了在中华绒螯蟹中进行SPF育苗的有效方法。
本发明的技术方案如下：
一种中华绒螯蟹活体无损取样方法，包括以下步骤：
步骤1，挑选中华绒螯蟹抱卵亲本，确保附肢齐全；
步骤2，固定螯足，对每个抱卵蟹在螯足底部进行套环标记，对标记好的每个个体在抱卵蟹腹部抱卵区采集不同发育期的受精卵于一个采样管中，得到取样样品。
进一步地，所述的中华绒螯蟹活体无损取样方法，步骤1中挑选中华绒螯蟹抱卵亲本选择个体健壮，生长良好的中华绒螯蟹。
进一步地，所述的中华绒螯蟹活体无损取样方法，步骤1中采集不同发育期的受精卵的采集体积为1-2mm3（30-50粒）。
进一步地，所述的中华绒螯蟹活体无损取样方法，步骤2中对每个抱卵蟹在螯足底部进行套环标记采用带数字编号的自锁式尼龙扎带进行。
进一步地，所述的中华绒螯蟹活体无损取样方法，步骤2中采样管为Eppendorf管。
所述的中华绒螯蟹活体无损取样方法得到的样品在中华绒螯蟹SPF育苗中的应用。
进一步地，所述的应用，中华绒螯蟹SPF育苗包括以下步骤：
步骤1，对取样样品进行DNA或RNA提取， 用PCR方法或LAMP方法进行目标病原的分子检测，确定抱卵蟹是否携带目标病原；
步骤2，对检测出不携带目标病原的抱卵蟹，进行隔离培育、孵化，幼体培育，获得SPF中华绒螯蟹大眼幼体。
以上所述的中华绒螯蟹活体无损取样方法在克氏原螯虾、日本沼虾或罗氏沼虾的活体取样中的应用。
利用本发明提供的活体无损取样方法得到的样品可以立即进行进行目标病原的分子检测实验；如果不立即检测，则可以速冻于液氮中带回实验室-80度冰箱保存待用。
与现有技术相比，本发明具有以下的优点：
本发明提供的活体无损取样方法简易可行而不伤抱卵蟹母体。SPF育苗要求亲本不带特定病原，必须首先对亲本进行检测，对经济甲壳类繁育亲本进行活体取样用于分子检测时，通常采用断肢取肌肉的方法，此法造成取样目标个体的伤残，活力下降，部分个体因为感染死亡，因此，该法不适用于需要长时间抱卵的部分虾蟹种类的SPF育苗的病原检测。而如果要检测其他组织，如肝胰腺、精卵巢等需要杀死检测亲本，更不可行。
中华绒螯蟹抱卵量通常在20-80万粒，受精卵附着于腹部附肢的刚毛上，卵粒圆形，直径0.3mm左右，受精卵易于采集且不损伤中华绒螯蟹母体，保证中华绒螯蟹亲蟹的正常抱卵孵幼。由于受精卵孵化为幼体，直接检测受精卵相比于检测亲蟹结果更为可信。通过检测受精卵可以做到确保后代幼体没有带特定病原。
本发明解决了中华绒螯蟹SPF育苗中活体无损取样检测的难题，为中华绒螯蟹SPF育苗提供了一条高效可行的途径，可望由此培育SPF健康苗种，提高中华绒螯蟹养殖经济效益。
具体实施方式：
实施例1
1. 2016年12月1日于江苏省射阳诚信生态育苗场，在一个2亩的池塘共有600只平均规格3.2两的母蟹，300只平均规格4.5两的公蟹，注入海水刺激进行交配,2016年12月20日抽干海水起捕，共获得抱卵蟹497只；
2. 对获得的497只抱卵蟹用带有数字编号的可以收紧的塑料套环扣在中华绒螯蟹大螯基部，对每个抱卵蟹进行编号；暂养于底部铺泥的水泥池中；
3. 对抱卵蟹进行活体无损采样：对附着于中华绒螯蟹母体腹部体外附肢的受精卵，用消毒干净镊子夹取2mm3受精卵（约50粒）分置于2个已编号的Eppendorf管中。
4. 对获得的中华绒螯蟹受精卵样品进行检测：对采集的每个抱卵蟹的2份受精卵样品， 1份样品用于DNA提取，提取DNA的方法采用北京康为世纪生物科技有限公司的SoilGenomic DNA Kit；另1份样品用于RNA提取，用Trizol试剂提取获得RNA，对获得的RNA用北京康为世纪生物科技有限公司的HiFiScript cDNA Synthesis Kit进行反转录获得cDNA。
以cDNA为模板进行EsRV（816，905，2012），EsBV进行PCR检测，以DNA为模板进行WSSV检测。用于PCR检测的病毒引物对序列如下：EsRV816（F: AGCGACCCGTTCACGCTTTA(SEQ IDNO.1)，R: TGACGCTGTGGAAGGACACG(SEQ ID NO.2)），EsRV905（F: CGCGTGAAGTTAGGGGTGGC(SEQ ID NO.3)，R: CGCCAACGCATCGTGAGACA (SEQ ID NO.4)），EsRV2012（F:AGCCAAAGGTGCCAGTAGAG(SEQ ID NO.5)，R: GCGAACACTTTCTCGCTGTC (SEQ ID NO.6)），EsBV（F: ACACCTGTGAGAGAGGTCTTGC(SEQ ID NO.7)，R: GTCTGCTGAGCAACTAGCCATTCT(SEQ IDNO.8)），WSSV（F: GCACTCGAAGTGACTGTGGGG(SEQ ID NO.9)，R: ATTGGTGCGCCAAAGGTGGTA(SEQ ID NO.10)），PCR反应体系是25ul：10×PCR Buffer 2.5ul，2.5mmol/L dNTP 0.5ul，MgCl2 1.5ul，每组反应体系两对引物的上下游引物各1ul，Taq酶0.3ul，DNA模板3ul，超纯水13.2ul。PCR反应程序为：94℃预变性3min; 94℃变性30s，58℃退火30s，72℃延伸30s，30个循环； 72℃延伸10min； 4℃保存。把PCR扩增产物在1.5%琼脂糖凝胶上进行电泳分离检测。最终通过检测获得402个不带目标病原的抱卵蟹；
5. SPF抱卵蟹隔离培育育苗：对获得的402个不带目标病原的抱卵蟹用消毒干净土池隔离培育；2017年4月5号，单独土池孵幼，最终于2017年5月6日获得SPF中华绒螯蟹大眼幼体共400斤，获得的中华绒螯蟹大眼幼体经抽样检测都不带所检测的5种病毒。
序列表
<110> 中国水产科学研究院淡水渔业研究中心
<120> 一种中华绒螯蟹活体无损取样方法及应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
agcgacccgt tcacgcttta 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tgacgctgtg gaaggacacg 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgcgtgaagt taggggtggc 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgccaacgca tcgtgagaca 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agccaaaggt gccagtagag 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcgaacactt tctcgctgtc 20
<210> 7
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
acacctgtga gagaggtctt gc 22
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtctgctgag caactagcca ttct 24
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gcactcgaag tgactgtggg g 21
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
attggtgcgc caaaggtggt a 21
Claims (8)
1.一种中华绒螯蟹活体无损取样方法，其特征在于，包括以下步骤：
步骤1，挑选中华绒螯蟹抱卵亲本，确保附肢齐全；
步骤2，固定螯足，对每个抱卵蟹在螯足底部进行套环标记，对标记好的每个个体在抱卵蟹腹部抱卵区采集不同发育期的受精卵于一个采样管中，得到取样样品。
2.根据权利要求1所述的中华绒螯蟹活体无损取样方法，其特征在于，步骤1中挑选中华绒螯蟹抱卵亲本选择个体健壮，生长良好的中华绒螯蟹。
3.根据权利要求1所述的中华绒螯蟹活体无损取样方法，其特征在于，步骤1中采集不同发育期的受精卵的采集体积为1-2mm3。
4.根据权利要求1所述的中华绒螯蟹活体无损取样方法，其特征在于，步骤2中对每个抱卵蟹在螯足底部进行套环标记采用带数字编号的自锁式尼龙扎带进行。
5.根据权利要求1所述的中华绒螯蟹活体无损取样方法，其特征在于，步骤2中采样管为Eppendorf管。
6.权利要求1所述的中华绒螯蟹活体无损取样方法得到的样品在中华绒螯蟹SPF育苗中的应用。
7.根据权利要求6所述的应用，其特征在于，中华绒螯蟹SPF育苗包括以下步骤：
步骤1，对取样样品进行DNA或RNA提取， 用PCR方法或LAMP方法进行目标病原的分子检测，确定抱卵蟹是否携带目标病原；
步骤2，对检测出不携带目标病原的抱卵蟹，进行隔离培育、孵化，幼体培育，获得SPF中华绒螯蟹大眼幼体。
8.权利要求1所述的中华绒螯蟹活体无损取样方法在克氏原螯虾、日本沼虾或罗氏沼虾的活体取样中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810184026.4A CN108432675B (zh) | 2018-03-06 | 2018-03-06 | 一种中华绒螯蟹活体无损取样方法的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810184026.4A CN108432675B (zh) | 2018-03-06 | 2018-03-06 | 一种中华绒螯蟹活体无损取样方法的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108432675A true CN108432675A (zh) | 2018-08-24 |
| CN108432675B CN108432675B (zh) | 2021-06-01 |
Family
ID=63193422
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810184026.4A Active CN108432675B (zh) | 2018-03-06 | 2018-03-06 | 一种中华绒螯蟹活体无损取样方法的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108432675B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295259A (zh) * | 2018-11-02 | 2019-02-01 | 中国水产科学研究院淡水渔业研究中心 | 一种基于rpa技术用于检测河蟹布尼亚病毒的引物及检测方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1817116A (zh) * | 2006-03-16 | 2006-08-16 | 王吉桥 | 中华绒螯蟹离体孵化育苗技术 |
| CN101213950A (zh) * | 2008-01-16 | 2008-07-09 | 淮阴师范学院 | 克氏原螯虾卵离体培育技术 |
| CN106172138A (zh) * | 2016-08-02 | 2016-12-07 | 盘锦光合蟹业有限公司 | 一种河蟹选育方法 |
| CN106922583A (zh) * | 2017-01-20 | 2017-07-07 | 扬州市嘉丰罗氏沼虾良种繁殖有限公司 | 一种罗氏沼虾spf（无病毒）苗种繁育方法 |
-
2018
- 2018-03-06 CN CN201810184026.4A patent/CN108432675B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1817116A (zh) * | 2006-03-16 | 2006-08-16 | 王吉桥 | 中华绒螯蟹离体孵化育苗技术 |
| CN101213950A (zh) * | 2008-01-16 | 2008-07-09 | 淮阴师范学院 | 克氏原螯虾卵离体培育技术 |
| CN106172138A (zh) * | 2016-08-02 | 2016-12-07 | 盘锦光合蟹业有限公司 | 一种河蟹选育方法 |
| CN106922583A (zh) * | 2017-01-20 | 2017-07-07 | 扬州市嘉丰罗氏沼虾良种繁殖有限公司 | 一种罗氏沼虾spf（无病毒）苗种繁育方法 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295259A (zh) * | 2018-11-02 | 2019-02-01 | 中国水产科学研究院淡水渔业研究中心 | 一种基于rpa技术用于检测河蟹布尼亚病毒的引物及检测方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108432675B (zh) | 2021-06-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kim et al. | Emergence and resurgence of the viral hemorrhagic septicemia virus (Novirhabdovirus, Rhabdoviridae, Mononegavirales) | |
| Anderson et al. | Molecular data implicate bryozoans as hosts for PKX (Phylum Myxozoa) and identify a clade of bryozoan parasites within the Myxozoa | |
| Timm et al. | Clitellata: oligochaeta | |
| CN101911918B (zh) | 牙鲆抗病优良品系选育方法 | |
| WO2015166408A2 (en) | Method for improving the production of cultured aquatic animals in combined rice-aquaculture systems | |
| Mulei et al. | Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates | |
| CN102007882A (zh) | 优质南美白对虾亲虾的繁育方法 | |
| Gadd et al. | Epidemiological aspects of viral haemorrhagic septicaemia virus genotype II isolated from Baltic herring, Clupea harengus membras L. | |
| CN105602945B (zh) | 一种同时检测海水鱼类三种致病菌的检测引物组、检测试剂盒和检测方法 | |
| Gadd et al. | Viral haemorrhagic septicaemia virus (VHSV) genotype II isolated from European river lamprey Lampetra fluviatilis in Finland during surveillance from 1999 to 2008 | |
| Liu et al. | Thelohanellus testudineus n. sp.(M yxosporea: B ivalvulida) infecting the skin of allogynogenetic gibel carp C arassius auratus gibelio (B loch) in C hina | |
| Kitamura et al. | Seasonal change of infective state of marine birnavirus in Japanese pearl oyster Pinctada fucata | |
| CN108179200A (zh) | 一种与克氏原螯虾高繁殖力性状连锁的微卫星标记及应用 | |
| Gómez et al. | Dinoflagellates Amyloodinium and Ichthyodinium (Dinophyceae), parasites of marine fishes in the South Atlantic Ocean | |
| Afanasyev et al. | Finding of Oriental River Prawn Macrobrachium nipponense (De Haan, 1849) in the Lower Terek River (Caspian Sea Basin) | |
| Eriksson-Kallio et al. | Susceptibility of rainbow trout to three different genogroups of infectious pancreatic necrosis virus | |
| Renault | Viruses infecting marine molluscs | |
| CN108432675A (zh) | 一种中华绒螯蟹活体无损取样方法及应用 | |
| CN103947607B (zh) | 一种草鱼抗细菌性败血症品系的构建方法 | |
| CN104396812A (zh) | 一种通过抑制受精卵极体释放获得异源多倍体泥鳅的方法 | |
| Gao et al. | Myxobolus jialingensis n. sp.(Myxozoa: Myxobolidae) infecting urinary bladder and hepatopancreas of yellowhead catfish Tachysurus fulvidraco from China | |
| Bigarré et al. | Ranaviruses associated with high mortalities in catfish in France | |
| CN103966316B (zh) | 一种二倍体泥鳅微卫星家系鉴定方法及其应用 | |
| Boyett | The ecology and microbiology of black band disease and brown band syndrome on the Great Barrier Reef | |
| Jebram et al. | New victorellids (Bryozoa, Ctenostomata) from North America: the use of parallel cultures in bryozoan taxonomy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |