CN112522422A - 一种基于coi基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法 - Google Patents
一种基于coi基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法 Download PDFInfo
- Publication number
- CN112522422A CN112522422A CN202011471296.7A CN202011471296A CN112522422A CN 112522422 A CN112522422 A CN 112522422A CN 202011471296 A CN202011471296 A CN 202011471296A CN 112522422 A CN112522422 A CN 112522422A
- Authority
- CN
- China
- Prior art keywords
- pelagic
- fish
- fishes
- coi gene
- scale
- 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.)
- Pending
Links
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 80
- 101150087323 COI gene Proteins 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims abstract description 14
- 239000012634 fragment Substances 0.000 title description 6
- 238000012408 PCR amplification Methods 0.000 claims abstract description 14
- 230000002068 genetic effect Effects 0.000 claims abstract description 7
- 241000876437 Brachymystax lenok Species 0.000 claims abstract description 6
- 230000004544 DNA amplification Effects 0.000 claims abstract description 6
- 238000012163 sequencing technique Methods 0.000 claims abstract description 6
- 101000921808 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) Probable cytochrome oxidase subunit 1 Proteins 0.000 claims abstract description 4
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 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 5
- 108010010803 Gelatin Proteins 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000012154 double-distilled water Substances 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 241000656145 Thyrsites atun Species 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 13
- 241000894007 species Species 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 4
- 108020005196 Mitochondrial DNA Proteins 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- 241000269908 Platichthys flesus Species 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000252206 Cypriniformes Species 0.000 description 1
- 101000919849 Homo sapiens Cytochrome c oxidase subunit 1 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种基于COI基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法。该方法包含:(1)分别提取飘鱼和寡鳞飘鱼基因组DNA;(2)PCR扩增飘鱼和寡鳞飘鱼的细胞色素氧化酶亚基I基因;(3)扩增产物纯化后,直接测序,飘鱼的COI基因扩增产物如SEQ ID NO.1所示,寡鳞飘鱼的COI基因扩增产物如SEQ ID NO.2所示;COI基因第4位点第6位点碱基分别是T和A为飘鱼,COI基因第4位点第6位点碱基分别是C和G为寡鳞飘鱼。该方法可以快速、简便、准确、有效地鉴别飘鱼和寡鳞飘鱼,结果稳定性好,重复率高,填补了目前国内分子生物学标准鉴别飘鱼和寡鳞飘鱼的空白,也有利于开展飘鱼和寡鳞飘遗传多样性研究和保护。
Description
技术领域
本发明属于分子遗传学领域,涉及一种基于COI基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法。
背景技术
飘鱼(Pseudolaubuca sinensis)和寡鳞飘鱼(Pseudolaubuca engraulis)隶属于鲤形目鲌亚科飘鱼属,是一类繁殖快、生活力强的小型鱼类,可食用,具有一定的经济价值。飘鱼和寡鳞飘鱼在我国分布广泛,从南至北诸河流及湖泊均有分布,喜成群于浅水区表层水面上来往漂游,杂食性,是淡水鱼类群落中的常见种类,数量较多,在水生态系统中具有重要的生态作用。多年来,受过度捕捞、水利工程、环境污染等不利因素的影响,飘鱼和寡鳞飘鱼种群数量下降,遗传多样性水平降低。飘鱼和寡鳞飘鱼形态相似,生态位相近,在鱼类资源调查中很难区分和鉴别。线粒DNA(mtDNA)为细胞核外遗传物质,具有母性遗传、结构简单、不发生重组、进化速度等特点。mtDNA在种间、种内群体间具有广泛的多态性,事研究动物物种间进化、进行种属鉴定的常用分子标记。线粒体细胞色素C氧化酶I基因(COI)是蛋白质编码基因之一,在鱼类遗传多样性、系统进化、种类鉴别等研究中具有重要用途,能够有效鉴别和区分近缘物种,现广泛用于分子鉴别领域。
发明内容
本发明的目的是针对形态区分鉴别的困难,提供一种飘鱼和寡鳞飘鱼的分子遗传鉴别方法。
本发明的目的通过以下技术方案实现:一种飘鱼和寡鳞飘鱼的分子遗传鉴别方法,包含以下步骤:(1)分别提取待鉴定的飘鱼和寡鳞飘鱼基因组DNA;(2)以提取的基因组DNA为模板,PCR扩增飘鱼和寡鳞飘鱼的细胞色素氧化酶亚基I基因,PCR扩增上游引物如序列SEQ ID NO.1所示,下游引物如序列SEQ ID NO.2所示;(3)扩增产物纯化后,直接测序,飘鱼的COI基因扩增产物如SEQ ID NO.3所示,寡鳞飘鱼的COI基因扩增产物如SEQ ID NO.4所示;COI基因第4位点第6位点碱基分别是T和A为飘鱼,COI基因第4位点第6位点碱基分别是C和G为寡鳞飘鱼。
SEQ ID NO.3:
GAATTAAGCCAACCTGGATCACTTCTGGGCGACGATCAAATTTATAACGTTATTGTTACTGCCCATGCCTTCGTAATAATTTTCTTTATAGTAATACCAATTCTTATTGGGGGGTTTGGAAACTGACTCGTGCCATTAATGATTGGGGCACCGGACATAGCATTCCCACGAATAAATAACATAAGCTTTTGACTTCTACCTCCCTCTTTCCTCCTACTATTAGCCTCTTCTGGCGTTGAAGCCGGGGCCGGTACTGGATGAACAGTTTACCCGCCACTTGCAGGCAACCTTGCTCATGCAGGGGCATCCGTAGACCTAACAATTTTCTCACTTCACCTAGCAGGTGTATCATCAATTTTAGGGGCAATTAACTTCATTACCACAACCATTAACATGAAACCACCAGCCATCTCTCAATACCAAACACCCCTATTTGTCTGAGCTGTACTTGTGACAGCCGTGCTCCTTCTTCTATCCCTACCAGTGCTAGCTGCTGGAATTACAATGCTCCTTACAGATCGAAACTTAAATACTACATTCTTTGATCCGGCGGGGGGAGGAGACCCGATCTTATACCAACACCTATTCTGATTCTTCGGC。
SEQ ID NO.4:
GAACTGAGTCAACCGGGATCACTTCTGGGCGACGATCAAATTTATAATGTTATTGTTACTGCCCATGCCTTCGTAATAATTTTCTTTATAGTAATGCCAATTCTTATCGGAGGGTTTGGAAACTGACTCGTGCCACTAATGATTGGGGCACCGGACATAGCCTTCCCCCGAATAAATAATATAAGCTTCTGACTCCTACCCCCCTCTTTCCTCCTGCTCCTAGCCTCCTCTGGCGTTGAGGCCGGAGCCGGTACAGGATGAACAGTCTACCCCCCACTCGCAGGCAATCTTGCCCATGCAGGAGCATCCGTAGACCTAACAATCTTCTCACTTCACTTAGCAGGTGTGTCCTCAATTTTAGGCGCAATTAATTTTATCACCACAACCATTAATATGAAACCACCAGCCATTTCTCAGTACCAAACACCTCTATTTGTTTGAGCCGTACTTGTAACAGCCGTACTTCTTCTCCTGTCCCTACCAGTCTTAGCGGCTGGAATTACAATGCTCCTTACAGACCGAAACCTAAATACTACCTTCTTTGATCCGGCAGGGGGAGGAGACCCAATCTTATACCAACACTTATTCTGATTCTTCGGC。
所述的PCR的反应体系为50 mL:80ng/μL的模板DNA 1 mL,PCR缓冲液 5 mL,dNTP混合液4 mL,每种dNTP 0.1mmol/L, 10 μmol/L的上、下游引物各1 mL,2mL 2.5 IU的Taq酶;双蒸水36mL;所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%(g/100ml)明胶组成;PCR扩增反应程序为:94 ℃预变性4 min,94 ℃变性40s,57 ℃退火50 s,72 ℃延伸60 s,经30个循环后再72 ℃延伸10 min。
作为本发明的另一方面,本发明提供一种用于飘鱼和寡鳞飘鱼的分子鉴别的引物对,其中:上游引物如序列SEQ ID NO.1所示,下游引物如序列SEQ ID NO.2所示。
作为本发明的另一方面,本发明提供所述的引物对在分子鉴别飘鱼和寡鳞飘鱼中的应用。
作为本发明的另一方面,本发明提供所述的引物对在制备飘鱼和寡鳞飘鱼分子鉴别试剂中的应用。
作为本发明的另一方面,本发明提供一种飘鱼和寡鳞飘鱼分子鉴别试剂,包含本发明所述的引物对。
优选的,所述的飘鱼和寡鳞飘鱼分子鉴别试剂还优选包括PCR缓冲液和dNTP混合液,所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%明胶组成。
有益效果:
本发明针对飘鱼和寡鳞飘鱼细胞色素氧化酶亚基I(Cytochrome Oxidasesubunit I,COI)基因序列差异,首次以飘鱼和寡鳞飘鱼的COI基因序列片段作为依据,从而定性鉴别飘鱼和寡鳞飘鱼。该方法可以快速、简便、准确、有效地鉴别飘鱼和寡鳞飘鱼,结果稳定性好,重复率高,填补了目前国内分子生物学标准鉴别飘鱼和寡鳞飘鱼的空白,也有利于开展飘鱼和寡鳞飘鱼物种多样性和遗传多样性研究。
附图说明
图1为本发明中COI基因测序序列比对结果,相同的碱基用连接号标识,差异的碱基用星号标识。飘鱼和寡鳞飘鱼从第4位点(COI-4)开始,碱基序列出现差异,其中飘鱼第4位点(COI-4)和第6位点(COI-6)碱基分别是T和A,30尾寡鳞飘鱼第4位点(COI-4)和第6位点(COI-6)碱基分别是C和G。
具体实施方式
以下结合实施例来进一步阐明本发明,但并不是对本发明做任何形式的限定,仅仅作示例说明。
实施例1
1、PCR引物
设计一对特异性PCR扩增引物,引物序列为:F:5’-TCGACTAATCATAAAGATATCGGCAC-3’(SEQ ID NO.1),下游引物为R:5’-ACTTCAGGGTGACCGAAGAATCAGAA-3’(SEQ ID NO.2)。
2、样本采集
从滆湖采集飘鱼和寡鳞飘鱼共60尾,其中飘鱼和寡鳞飘鱼个体各30尾。
3、基因组DNA提取
取每尾鱼的肌肉组织用于基因组DNA提取。采用Takara公司的光谱性基因组提取试剂盒提取DNA,用去离子水溶解DNA,紫外分光光度计测定DNA溶液OD值并稀释至80ng/μL,-20℃保存备用。
4、PCR扩增与检测
已提取的DNA为模板,用上述引物(F和R)进行PCR扩增。PCR反应体系为50 mL:1 mL模板DNA(800ng/μL);PCR缓冲液 5 mL(10mmol/L Tris-HCl,pH9.0,0.5mmol/L KCl,30mmol/L MgCl2,0.01%明胶);dNTP混合液4 mL (每种dNTP 0.1mmol/L);上、下游引物(10μmol/L)各1 mL,2mL Taq酶(2.5 IU);双蒸水36mL。扩增反应程序为:94 ℃预变性4 min,94℃变性40 s,57℃退火50 s,72 ℃延伸60 s,经30个循环后再72 ℃延伸10 min。
PCR产物在1.5 %琼脂糖凝胶电泳中检测分离,120 V电压下电泳30 min之后,经凝胶成像分析系统确定 PCR 扩增产物的大小及纯度。
5、测序比对
检测后的PCR扩增产物直接送至生物公司纯化测序,获得COI基因片段,经分析获得长度为600bp的COI基因序列片段。经多重比对后可以发现COI基因从第4位点(COI-4)的碱基序列存在差异:30尾飘鱼第4位点(COI-4)和第6位点(COI-6)碱基分别是T和A,30尾寡鳞飘鱼第4位点(COI-4)和第6位点(COI-6)碱基分别是C和G。
实施例2
按照实施例1方法,从太湖、高邮湖、洪泽湖、骆马湖和庆安水库采集样本,将样本扩大到飘鱼和寡鳞飘鱼个体各80尾,结果依然显示飘鱼COI基因第4位点(COI-4)和第6位点(COI-6)碱基分别是T和A,30尾寡鳞飘鱼第4位点(COI-4)和第6位点(COI-6)碱基分别是C和G。
序列表
<110> 江苏省淡水水产研究所
<120> 一种基于COI基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法
<141> 2020-12-01
<160> 4
<170> SIPOSequenceListing 1.0
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 3
tcgactaatc ataaagatat cggcac 26
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 2
acttcagggt gaccgaagaa tcagaa 26
<210> 3
<211> 600
<212> DNA
<213> Artificial Sequence
<400> 3
gaattaagcc aacctggatc acttctgggc gacgatcaaa tttataacgt tattgttact 60
gcccatgcct tcgtaataat tttctttata gtaataccaa ttcttattgg ggggtttgga 120
aactgactcg tgccattaat gattggggca ccggacatag cattcccacg aataaataac 180
ataagctttt gacttctacc tccctctttc ctcctactat tagcctcttc tggcgttgaa 240
gccggggccg gtactggatg aacagtttac ccgccacttg caggcaacct tgctcatgca 300
ggggcatccg tagacctaac aattttctca cttcacctag caggtgtatc atcaatttta 360
ggggcaatta acttcattac cacaaccatt aacatgaaac caccagccat ctctcaatac 420
caaacacccc tatttgtctg agctgtactt gtgacagccg tgctccttct tctatcccta 480
ccagtgctag ctgctggaat tacaatgctc cttacagatc gaaacttaaa tactacattc 540
tttgatccgg cggggggagg agacccgatc ttataccaac acctattctg attcttcggc 600
<210> 4
<211> 600
<212> DNA
<213> Artificial Sequence
<400> 4
gaactgagtc aaccgggatc acttctgggc gacgatcaaa tttataatgt tattgttact 60
gcccatgcct tcgtaataat tttctttata gtaatgccaa ttcttatcgg agggtttgga 120
aactgactcg tgccactaat gattggggca ccggacatag ccttcccccg aataaataat 180
ataagcttct gactcctacc cccctctttc ctcctgctcc tagcctcctc tggcgttgag 240
gccggagccg gtacaggatg aacagtctac cccccactcg caggcaatct tgcccatgca 300
ggagcatccg tagacctaac aatcttctca cttcacttag caggtgtgtc ctcaatttta 360
ggcgcaatta attttatcac cacaaccatt aatatgaaac caccagccat ttctcagtac 420
caaacacctc tatttgtttg agccgtactt gtaacagccg tacttcttct cctgtcccta 480
ccagtcttag cggctggaat tacaatgctc cttacagacc gaaacctaaa tactaccttc 540
tttgatccgg cagggggagg agacccaatc ttataccaac acttattctg attcttcggc 600
Claims (7)
1.一种飘鱼和寡鳞飘鱼的分子鉴别方法,其特征在于:包含以下步骤,
(1)分别提取待鉴定的飘鱼和寡鳞飘鱼基因组DNA;
(2)以提取的基因组DNA为模板,PCR扩增飘鱼和寡鳞飘鱼的细胞色素氧化酶亚基I基因,PCR扩增上游引物如序列SEQ ID NO.1所示,下游引物如序列SEQ ID NO.2所示;
(3)扩增产物纯化后,直接测序,飘鱼的COI基因扩增产物如SEQ ID NO.3所示,寡鳞飘鱼的COI基因扩增产物如SEQ ID NO.4所示;COI基因第4位点第6位点碱基分别是T和A为飘鱼,COI基因第4位点第6位点碱基分别是C和G为寡鳞飘鱼。
2.根据权利要求1所述的分子鉴别方法,其特征在于:所述的PCR的反应体系为50 mL:80ng/μL的模板DNA 1 mL,PCR缓冲液 5 mL,dNTP混合液4 mL,每种dNTP 0.1mmol/L, 10 μmol/L的上、下游引物各1 mL,2mL 2.5 IU的Taq酶;双蒸水36mL;所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%(g/100ml)明胶组成;PCR扩增反应程序为:94 ℃预变性4 min,94 ℃变性40 s,57 ℃退火50 s,72 ℃延伸60 s,经30个循环后再72 ℃延伸10 min。
3.一种用于飘鱼和寡鳞飘鱼的分子鉴别的引物对,其特征在于:上游引物如序列SEQID NO.1所示,下游引物如序列SEQ ID NO.2所示。
4.权利要求3所述的引物对在分子遗传鉴别飘鱼和寡鳞飘鱼中的应用。
5.权利要求3所述的引物对在制备飘鱼和寡鳞飘鱼分子鉴别试剂中的应用。
6.一种飘鱼和寡鳞飘鱼分子鉴别试剂,其特征在于:包含权利要求3所述的引物对。
7.根据权利要求6所述的飘鱼和寡鳞飘鱼分子鉴别试剂,其特征在于:所述的飘鱼和寡鳞飘鱼分子鉴别试剂还包括PCR缓冲液和dNTP混合液,所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%明胶组成。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011471296.7A CN112522422A (zh) | 2020-12-14 | 2020-12-14 | 一种基于coi基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011471296.7A CN112522422A (zh) | 2020-12-14 | 2020-12-14 | 一种基于coi基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112522422A true CN112522422A (zh) | 2021-03-19 |
Family
ID=74999692
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011471296.7A Pending CN112522422A (zh) | 2020-12-14 | 2020-12-14 | 一种基于coi基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112522422A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114836414A (zh) * | 2022-04-15 | 2022-08-02 | 江苏省淡水水产研究所 | 一种分子鉴别斑点叉尾鮰和云斑鮰的引物及方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109055401A (zh) * | 2018-07-25 | 2018-12-21 | 江汉大学 | 一种寡鳞飘鱼dna条形码序列及其应用 |
-
2020
- 2020-12-14 CN CN202011471296.7A patent/CN112522422A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109055401A (zh) * | 2018-07-25 | 2018-12-21 | 江汉大学 | 一种寡鳞飘鱼dna条形码序列及其应用 |
Non-Patent Citations (3)
Title |
---|
GENBANK: "KC429670.1", 《GENBANK》, 10 March 2014 (2014-03-10) * |
GENBANK: "MH664212.1", 《GENBANK》, 31 December 2019 (2019-12-31) * |
YANJUN SHEN等: "DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River", 《ECOLOGY AND EVOLUTION》, vol. 6, no. 9, 17 May 2016 (2016-05-17), pages 2 - 3 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114836414A (zh) * | 2022-04-15 | 2022-08-02 | 江苏省淡水水产研究所 | 一种分子鉴别斑点叉尾鮰和云斑鮰的引物及方法 |
CN114836414B (zh) * | 2022-04-15 | 2023-09-22 | 江苏省淡水水产研究所 | 一种分子鉴别斑点叉尾鮰和云斑鮰的引物及方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109943645B (zh) | 一种淡水鱼类线粒体12s通用宏条形码扩增引物及其应用方法 | |
CN114686597A (zh) | 一种银龙鱼性别鉴定snp分子标记及其应用 | |
CN110343767A (zh) | 凡纳滨对虾微卫星分子标记特异性引物及其在遗传多样性分析中的应用 | |
CN109880893B (zh) | 用于丝尾鱯性别鉴定的特异性dna片段及应用 | |
KR101499695B1 (ko) | 바리과 어류 중 능성어 및 붉바리 신속 판별 유전자 마커 및 판별방법 | |
CN100415884C (zh) | 一种用于研究鱼类遗传关系的dna分子标记方法 | |
WO2022068215A1 (zh) | 凡纳滨对虾抗弧菌相关est-str标记及其特异性引物和检测方法 | |
CN112522422A (zh) | 一种基于coi基因片段的飘鱼和寡鳞飘鱼的分子鉴别方法 | |
CN113637765A (zh) | 鉴定大口黑鲈遗传性别的分子标记及应用 | |
CN111073983B (zh) | 与大口黑鲈北方亚种和佛罗里达亚种鉴定相关的snp标记及其应用 | |
CN102321750A (zh) | 一种用分子标记快速筛选边鸡体重增长程度的方法 | |
CN110042169B (zh) | 一种黑龙江茴鱼群体特异性分子标记引物、试剂盒及鉴定方法 | |
CN110452992A (zh) | 一种凡纳滨对虾est微卫星位点引物的标记方法 | |
CN104099423A (zh) | 用于刀鲚不同生态型种群识别的分子标记 | |
CN110951892B (zh) | 用于几种鲟鱼物种鉴定的ssr引物对组、试剂盒、鉴定方法及应用 | |
CN112575092A (zh) | 一种基于Cytb基因序列的飘鱼和寡鳞飘鱼的分子鉴别方法 | |
CN114134237A (zh) | 鉴定大口黑鲈美国北方亚种和优鲈3号亲本的srap分子标记及其应用 | |
CN104073562B (zh) | 一种用于刀鲚不同生态型种群识别的分子标记 | |
CN111733256A (zh) | 一种基于Cytb基因片段的蛇鮈和长蛇鮈的分子鉴别方法 | |
CN110616270A (zh) | 一种基于coi基因序列的*和贝氏*的分子鉴别方法 | |
CN104651525B (zh) | 一种用于毛蚶家系鉴定的多重pcr方法 | |
CN108504751B (zh) | 微卫星标记鉴定鲫鱼倍性的方法及其应用 | |
CN102251051A (zh) | 一种检测边鸡mstn基因型的试剂盒 | |
KR102074139B1 (ko) | 미꾸리과 어종 판별용 유전자 마커 및 판별방법 | |
CN110846419B (zh) | 用于欧鳇和闪光鲟物种鉴定的ssr引物对组、试剂盒、鉴定方法及应用 |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210319 |