CN107557484A - A kind of Kumamoto oyster based on DNA specific fragments（Crassostreasikamea）Authentication method - Google Patents
A kind of Kumamoto oyster based on DNA specific fragments（Crassostreasikamea）Authentication method Download PDFInfo
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- CN107557484A CN107557484A CN201711041921.2A CN201711041921A CN107557484A CN 107557484 A CN107557484 A CN 107557484A CN 201711041921 A CN201711041921 A CN 201711041921A CN 107557484 A CN107557484 A CN 107557484A
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Abstract
The invention belongs to aquatic animal Germplasm Identification field, is related to a kind of molecular biology method that Kumamoto oyster species based on DNA specific fragments differentiate, brand-new, efficiently, can specifically identify Kumamoto oyster from widely distributed oyster.Method provided by the invention, comprises the following steps：(1) oyster genomic DNA is extracted；(2) enter performing PCR with the oyster genomic DNA in primer pair step (1) to expand, obtain the PCR primer of purpose fragment；Wherein, sense primer is Cs F2：5` CGAAGAGGGGCATGATAAATGAGG 3`, anti-sense primer are Cs R2：5`‑ATATGAACTTCTCCAACCTCCCC‑3`；(3) PCR primer in step (2) is subjected to gel electrophoresis, the as Kumamoto oyster of signal band occurs in 100bp.The beneficial effects of the invention are as follows the discriminating of carry out Kumamoto oyster that can be economic, quick, easy, the accuracy rate that the result display of sequence verification differentiates is 100%.
Description
Technical field
This patent belongs to aquatic animal Germplasm Identification field, is related to a kind of Kumamoto oyster based on DNA specific fragments
Molecular biology method that species differentiate, brand-new, efficiently, it can specifically identify Kumamoto from widely distributed oyster
Oyster.
Background technology
Coastal area of china oyster aboundresources, the suitable reef oyster of making of the various selection of species is the important life in ecology
Topic.Oyster (Ostreidae) is worldwide wide topological classes, is commonly called as oyster, and alias oyster is yellow, oyster is white, oyster.Its meat flavour is delicious,
Soft texture is delicate, and nutritious, European people claims oyster to be " Yemana of ocean ", and " milk of ocean ", roman praises it
For " marine delicious holy fish ".Japanese is then called " source of root ", " the super rice of ocean ".Oyster is first to cultivate shellfish greatly in the world
Class, kind more than 100 is shared, common are 5 kinds in China coast, i.e.,:Kumamoto oyster (Crassostreasikamea), Crassostrea rivularis
(C.ariakensis), long oyster (C.gigas), the huge oyster in Hong Kong (C.hongkongensis), Portuguese oyster
(C.angulata).Oyster bioherm (oyster reef) is to assemble the one kind for being grown on hard substrate surface and being formed by a large amount of oysters
Organic reef system, it is distributed widely in temperate zone river mouth and littoral area.It is male except providing a large amount of fresh and alive oysters for the mankind with addition to edible
Oyster reef also there are highly important ecological functions to be worth with environmental services.Oyster is the oyster bioherm ecosystem " foundation stone species
(keystone species)”.It is reported that in all oysters of coastal area of china distribution, there are the species for making reef ability to have nearly river
Oyster, long oyster and Kumamoto oyster；And Portuguese oyster and Hong Kong oyster are not yet reported at present.
It is accurate to differentiate oyster species (including adult and young), for analysing in depth the spatial niche of oyster and making reef energy
Power, oyster is examined to illustrate oyster bioherm habitat complexity and ecological functions to the competitive utilization and colonization ability in attachment substrate space
Between correlation, be all vital, the screening of target species in engineering can be recovered for oyster bioherm scientific guidance be provided.
Oyster species is not easily distinguishable from form.Oyster bioherm recovers to turn into maintenance offshore fishery resources, repairs offshore fishing
One of important means of industry aquatic ecosystem.Oyster (Ostreidae) is worldwide wide topological classes, because oyster is that battalion is solid
The mollusk of life, greatly change often occurs with the difference of living environment for its formalness, and most of species are simple
Formalness by shell is very indistinguishable.
The oyster young is difficult to identify.The oyster young differentiates even more difficult because similarity is big；And traditional morphology
Classification indicators, it is empirical, expert's dependent form mostly, for ordinary person, particularly grass-roots work person, uses not enough
It is convenient and practical.Therefore, in daily management and research, there is an urgent need to concise, quick, stable discrimination method.DNA is to lose
The carrier of information is passed, it is highly stable, it is not easy protected from environmental, is conventional spe cies identification mark.
Chinese patent CN 105734133A disclose a kind of Crassostrea rivularis based on mitochondria specific fragment and Kumamoto oyster
Authentication method.Including：Extract the genomic DNA of Crassostrea rivularis and Kumamoto oyster；Using sense primer and anti-sense primer to base
Because a group DNA enters performing PCR amplification, the PCR primer of purpose fragment is obtained；Wherein, sense primer 5 '-
GTTCCGTTCCATCCTTAT-3 ' ,-AGACCACTTATCCCTCCA-3 ' of anti-sense primer 5 '；PCR primer is directly coagulated
Gel electrophoresis, according to obtained PCR primer band and the result of sequencing is combined, to judge species classification.What the patent application was used
DNA purpose fragment is 1506 ± 2bp, it is impossible to which quick, easy distinguishes Kumamoto oyster and other 4 seed oysters, also finally
It is combined sequencing and carrys out judged result.
The content of the invention
In order to solve during oyster adult and the young differentiate, the problem of traditional morphological indexes are difficult to identification, base of the present invention
In oyster genome, develop DNA specific fragments, the specific fragment amplified using PCR method, be it is a kind of it is easy to operate, into
The molecular identification method of this low, time-consuming Kumamoto oyster short, accuracy rate is high.
Kumamoto oyster authentication method provided by the invention based on DNA specific fragments, comprises the following steps：
(1) oyster genomic DNA is extracted；
(2) enter performing PCR with the oyster genomic DNA in primer pair step (1) to expand, obtain the PCR primer of purpose fragment；
Wherein, sense primer Cs-F2：
5`-CGAAGAGGGGCATGATAAATGAGG-3`, anti-sense primer Cs-R2：
5`-ATATGAACTTCTCCAACCTCCCC-3`；
(3) PCR primer in step (2) is subjected to gel electrophoresis, the as Kumamoto oyster of signal band occurs in 100bp,
Other oysters do not have signal strips band.
Wherein, the method for extraction oyster genomic DNA is to be extracted to try with marine animal tissue gene group DNA in step (1)
Agent box extracts.
Wherein, the purpose fragment of Kumamoto oyster is 114bp in PCR primer band in step (2).
Wherein, the condition that PCR is expanded in step (2) is first 95 DEG C of pre-degeneration 3min, followed by 35 circulate, each circulation
Including 95 DEG C of denaturation 15sec, 55 DEG C of renaturation 15sec, 72 DEG C of extension 15sec, are finally 72 DEG C of extension 3min.
Wherein, the Ago-Gel that the condition of gel electrophoresis is 3% in step (3).
Wherein, the condition of gel electrophoresis is in step (3)：Voltage 200V, time 28min, buffer solution TBE.
The discriminating of carry out Kumamoto oyster that can be economic, quick, easy using the present invention, the result display mirror of sequence verification
Other accuracy rate is 100%；This authentication method will play weight in aquaculture, resource investigation, environmental protection, restoration of the ecosystem
Act on.
Compared with prior art, the present invention has the advantages that：
(1) method is simple and easy, it is not necessary to carries out examining order；
(2) can intuitively be detected according to agarose gel electrophoresis；
(3) this method is accurately feasible, is widely used, and oyster adult even can be examined to the oyster young in water body environment
Survey.
Brief description of the drawings
Fig. 1 is in mitochondria base after obtaining sense primer in embodiment 1 by engineer's primer and its compared with Mega7
Because of the position of group；
Fig. 2 is in mitochondria base after obtaining anti-sense primer in embodiment 1 by engineer's primer and its compared with Mega7
Because of the position of group；
Fig. 3 is PCR primer electrophoresis result figure in embodiment 1；Wherein, the 1st swimming lane is that marker, 2-5 swimming lane are bear from left to right
This oyster, 6-9 swimming lanes be Crassostrea rivularis 10-13 swimming lanes be Portuguese oyster, 14-17 swimming lanes are long oyster, 18-21 swimming lanes are fragrant
Port oyster, 22-25 swimming lanes are negative control.
Fig. 4 is PCR primer sequencing result.Each sequence is followed successively by from top to bottom：1-17 is Kumamoto oyster；20 be Kumamoto gene
Group.
Embodiment
With reference to embodiment, the invention will be further described：
Embodiment
1st, experiment material
In the main sea area (Ningbo of Zhejiang in China；Shanghai reed tidal harbour；Jiangsu Haimen；The Shandong Bohai Sea：Weihai, Dalian Bay；Fujian
Ningde；Guangdong Yangjiang) intertidal zone gather 5 seed oysters and tested, including Kumamoto oyster, Crassostrea rivularis, Portuguese oyster, length
Oyster, Hong Kong oyster.Collecting sample musculature, -20 DEG C save backup.
2nd, extracting genome DNA
Using the genomic DNA of marine animal tissue gene group DNA extraction kit extraction oyster.
3rd, PCR is expanded
Using oyster genomic DNA as template, the mitochondria of other oyster kinds occurred according to Kumamoto oyster and China coast
DNA sequence dna, by comparing design primer, (primer sequence is：Cs-F2：5`-CGAAGAGGGGCATGATAAATGAGG-3`, downstream
Primer is Cs-R2：5`-ATATGAACTTCTCCAACCTCCCC-3`), performing PCR amplification is entered.Primer location is shown in Fig. 1 and Fig. 2.
Wherein, it is with the purpose fragment PCR reaction conditions of the above-mentioned upstream and downstream primer amplification mitochondrial DNA of our designs：
First 95 DEG C of pre-degeneration 3min, followed by 35 circulations, each circulation include 95 DEG C and are denatured 15sec, 55 DEG C of renaturation 15sec, 72 DEG C
Extend 15sec, be finally 72 DEG C of extension 3min.
4th, PCR primer electrophoresis
PCR primer electrophoresis, deposition condition in 3% Ago-Gel are：Voltage 200V, time 28min, buffer solution are
TBE.As a result show determine to have at 100bp band for Kumamoto oyster (Fig. 3).Kumamoto oyster purpose fragment length is 114bp,
And other oyster kinds at 100bp without band.
5th, to the sequence verification of PCR primer
PCR primer is sequenced using Sanger dideoxy chain termination, then compared with Vector NTI softwares,
As a result (see Fig. 4) consistent with the design attitude of this primer is shown.
Described above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment
Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within
Enclose.
Sequence table
<110>Donghai Aquatic Products Inst., China Aquatic Science Research Academy
<120>A kind of Kumamoto oyster authentication method based on DNA specific fragments
<141> 2017-10-30
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial sequence
<400> 1
cgaagagggg catgataaat gagg 24
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence
<400> 2
atatgaactt ctccaacctc ccc 23
Claims (6)
1. a kind of Kumamoto oyster authentication method based on DNA specific fragments, it is characterised in that comprise the following steps：
(1) oyster genomic DNA is extracted；
(2) enter performing PCR with the oyster genomic DNA in primer pair step (1) to expand, obtain the PCR primer of purpose fragment；Its
In, sense primer Cs-F2：
5`-CGAAGAGGGGCATGATAAATGAGG-3`, anti-sense primer Cs-R2：
5`-ATATGAACTTCTCCAACCTCCCC-3`；
(3) PCR primer in step (2) is subjected to gel electrophoresis, the as Kumamoto oyster of signal band occurs in 100bp.
2. the molecular identification method of Kumamoto oyster according to claim 1, it is characterised in that extraction oyster in step (1)
The method of genomic DNA is to be extracted with marine animal tissue gene group DNA extraction kit.
3. the molecular identification method of Kumamoto oyster according to claim 1, it is characterised in that PCR primer bar in step (2)
The purpose fragment of Kumamoto oyster is 114bp in band.
4. the molecular identification method of Kumamoto oyster according to claim 1, it is characterised in that PCR is expanded in step (2)
Condition is first 95 DEG C of pre-degeneration 3min, followed by 35 circulations, and each circulation includes 95 DEG C of denaturation 15sec, 55 DEG C of renaturation
15sec, 72 DEG C of extension 15sec, it is finally 72 DEG C of extension 3min.
5. the molecular identification method of Kumamoto oyster according to claim 1, it is characterised in that gel electrophoresis in step (3)
Condition be 3% Ago-Gel.
6. the molecular identification method of Kumamoto oyster according to claim 1, it is characterised in that gel electrophoresis in step (3)
Condition be：Voltage 200V, time 28min, buffer solution TBE.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626238A (en) * | 2021-01-19 | 2021-04-09 | 浙江万里学院宁海海洋生物种业研究院 | Characteristic sequence for identifying crassostrea sikamea, specificity identification primer and identification method |
Citations (3)
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| CN101130815A (en) * | 2007-09-18 | 2008-02-27 | 中国科学院南海海洋研究所 | PCR-RFLP identification method for seven crassostrea oyster on south China coast |
| CN102758009A (en) * | 2012-06-08 | 2012-10-31 | 中国科学院海洋研究所 | Method for identifying related species of oyster |
| CN105734134A (en) * | 2016-03-22 | 2016-07-06 | 中国水产科学研究院东海水产研究所 | Method of molecularly identifying crassostrea rivularis and crassostrea sikamea |
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2017
- 2017-10-30 CN CN201711041921.2A patent/CN107557484A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130815A (en) * | 2007-09-18 | 2008-02-27 | 中国科学院南海海洋研究所 | PCR-RFLP identification method for seven crassostrea oyster on south China coast |
| CN102758009A (en) * | 2012-06-08 | 2012-10-31 | 中国科学院海洋研究所 | Method for identifying related species of oyster |
| CN105734134A (en) * | 2016-03-22 | 2016-07-06 | 中国水产科学研究院东海水产研究所 | Method of molecularly identifying crassostrea rivularis and crassostrea sikamea |
Non-Patent Citations (3)
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| WANG JIAFENG ET AL.: "A new identifi cation method for fi ve species of oysters in genus Crassostrea from China based on high-resolution melting analysis", 《CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626238A (en) * | 2021-01-19 | 2021-04-09 | 浙江万里学院宁海海洋生物种业研究院 | Characteristic sequence for identifying crassostrea sikamea, specificity identification primer and identification method |
| CN112626238B (en) * | 2021-01-19 | 2022-02-22 | 浙江万里学院宁海海洋生物种业研究院 | Characteristic sequence for identifying crassostrea sikamea, specificity identification primer and identification method |
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Application publication date: 20180109 |