CN102758009B - Method for identifying related species of oyster - Google Patents
Method for identifying related species of oyster Download PDFInfo
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- CN102758009B CN102758009B CN 201210189381 CN201210189381A CN102758009B CN 102758009 B CN102758009 B CN 102758009B CN 201210189381 CN201210189381 CN 201210189381 CN 201210189381 A CN201210189381 A CN 201210189381A CN 102758009 B CN102758009 B CN 102758009B
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- 241000237502 Ostreidae Species 0.000 title claims abstract description 48
- 235000020636 oyster Nutrition 0.000 title claims abstract description 40
- 241000894007 species Species 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000002844 melting Methods 0.000 claims abstract description 16
- 230000008018 melting Effects 0.000 claims abstract description 16
- 230000003321 amplification Effects 0.000 claims abstract description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 7
- 238000012408 PCR amplification Methods 0.000 claims description 15
- 101150087323 COI gene Proteins 0.000 claims description 10
- 230000004304 visual acuity Effects 0.000 claims description 9
- 238000013461 design Methods 0.000 claims description 8
- 238000011156 evaluation Methods 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 230000004069 differentiation Effects 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 235000015170 shellfish Nutrition 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 3
- 102000053602 DNA Human genes 0.000 abstract 2
- 108020004414 DNA Proteins 0.000 abstract 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract 2
- 241000196324 Embryophyta Species 0.000 description 14
- 230000008859 change Effects 0.000 description 8
- 238000011160 research Methods 0.000 description 6
- 238000004153 renaturation Methods 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 241000237519 Bivalvia Species 0.000 description 2
- 241000548230 Crassostrea angulata Species 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 241000358845 Crassostrea ariakensis Species 0.000 description 1
- 241001207609 Crassostrea hongkongensis Species 0.000 description 1
- 241000358847 Crassostrea sikamea Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000003466 welding Methods 0.000 description 1
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Abstract
The invention relates to a related specie identification technology, in particular to a method for identifying related species of an oyster. The method specifically comprises the following steps of: by using an oyster genome DNA (Deoxyribonucleic Acid) as a template, designing a primer according to a tag sequence of the oyster specie genome; carrying out PCR (Polymerase Chain Reaction) amplification on the primer; and distinguishing oyster species by using Tm value difference in a high-resolution melting curve of an amplification product. Through the adoption of the method provided by the invention, the species can be identified rapidly, economically, simply and conveniently; and compared with a tag sequence sequencing result, an identifying result of the method provided by the invention is 100% in accuracy.
Description
Technical field
The present invention relates to the sibling species authenticate technology, the method for the nearly edge species evaluation of a seed oyster specifically.
Background technology
Oyster belongs to the lamellibranchiata (or Bivalvia) in the Mollusca.Oyster has very high economic worth, is the important aquaculture objects in countries in the world, has become the economic animal of China and even world's aquaculture output maximum.Especially finishing of oyster genome sequencing indicates that oyster has entered the genome epoch, the molecular biology research of oyster, and functional gene is learned research, and group is learned research and is waited related basic research and action oriented research to launch in succession.The differentiation of species and evaluation are the bases of various research work, yet oyster species kind is very complicated, and profile very easily is subjected to the influence of environment; Also have many disputes and difference about its classification, classification foundation also is not quite similar.In recent years, along with development of molecular biology, utilize molecular biology method to identify that species have become a kind of trend gradually, and accepted by most biologist.At present modal is to use mitochondrial COI gene and ITS sequence difference as the classification foundation of distinguishing different plant species, but the difference between these gene orders is SNP or In/de l often, therefore the variation of sequence length and not obvious between the different plant species is difficult to use method difference different plant species such as electrophoresis; Order-checking can obtain the detailed base information of this gene or sequence, but the experimentation that checks order is more loaded down with trivial details, spend also higherly, and needs the support of large-scale instrument, be difficult to realize at short notice accurately, fast, simple species evaluation.
Summary of the invention
The object of the invention is to provide the seed oyster method that nearly edge species are identified.
For achieving the above object, the technical solution used in the present invention is:
The method that the nearly edge species of one seed oyster are identified, be template with the oyster genomic dna, sequence label design primer according to each species gene group of oyster carries out pcr amplification, utilize amplified production at high resolving power melting curve (High Resolution Melting, HRM) the different and then differentiation oyster species of the Tm value difference in.
Obtain the PCR product of 40bp-100bp by above-mentioned amplification, added behind the nucleic acid saturable dye 95 ℃ of denaturing treatment 10 minutes, be cooled to room temperature and carry out HRM and measure.
Be template with oyster genomic dna to be identified, carry out pcr amplification according to the COI gene order design primer of oyster, utilize the Tm value difference of amplified production in high resolving power melting curve (HRM) different and then distinguish the close species of shellfish; The sequence amplification primer is:
COI-F1：TACTTAATATTGGGTTTTTAGGGT；
COI-R1：CGCGTATCAATATCCATTCC。
The advantage that the present invention has:
1. method is simple; Do not need order-checking, directly come the difference on the reaction dna level to distinguish different plant species by the Tm value of amplified fragments.
2. the result is clearly directly perceived; Need not carry out steps such as sequence alignment, the difference of dna level shows with peak diagram form intuitively between the different plant species, very easily distinguishes and distinguishes.
3. accurate and effective; Authentication method of the present invention adopts high resolving power melting curve (HRM) to have the ability of differentiating single nucleotide difference, can accurately reflect the difference on the dna level, makes qualification result accurately and reliably, and the accuracy rate of comparing with sequencing result is 100%.
4. applicability is extensive; Different with order-checking, do not need all individualities to be identified sequencing analysis one by one.One group of primer can carry out the species evaluation and not need to redesign primer at unlimited how similar individuality among the present invention.Secondly, the present invention is an open system, has wide range of applications; The oyster species that can distinguish based on the difference on the dna level not only can be used for the evaluation of the nearly edge species of specific a few seed oyster, so long as all can utilize this method to identify.
Description of drawings
The huge oyster that Fig. 1 provides for the embodiment of the invention belong to the nearly edge species of oyster identify high resolving power melting curve figure (wherein X-coordinate be temperature (℃), ordinate zou is the logarithmic value of fluorescence intensity; The corresponding X-coordinate of the vertex of every curve is the Tm value of the PCR product of this curve correspondence).
The huge oyster that Fig. 2 provides for the embodiment of the invention belongs to the nearly edge species of oyster pcr amplification product 1% agarose gel electrophoresis detection figure.
Embodiment
The present invention carries out relying on the nucleotide sequence difference introducing high resolving power melting curve method in the process that species identify, only with one couple of PCR primers the difference of different plant species on dna level is changed into more intuitive melting curve.The steps include:
A. the acquisition of species appraisal basis sequence: from the data with existing storehouse or other source obtain to distinguish target species according to sequence (as biological sequence labels such as COI gene orders).
B. design of primers: between the comparison different plant species according to the difference of sequence, the zone of target species can be obviously distinguished in searching, utilize primer premier5 software in this zone, to design the pcr amplification primer, make amplified production that obvious sequence difference (being that the Tm value is different) should be arranged between different plant species, mainly be presented as G, C content difference.
The c.PCR amplification: the genomic dna with target shellfish to be identified is template, utilizes above-mentioned primer to carry out pcr amplification.
D. product detects and handles: utilize 1% agarose gel electrophoresis to detect above-mentioned pcr amplification product, determine that amplified production is single, behind the no non-specific amplification, 1 μ l nucleic acid saturable dye LC-green (Idaho is added in each reaction, the U.S.), behind 95 ℃ of sex change 10min of regular-PCR instrument operation, be cooled to room temperature.
E. high resolving power melting curve (HRM) is analyzed: above-mentioned product moves HRM on Lightscanner96 (Idaho, the U.S.) platform, utilizes the analysis software on the platform to obtain the product melting curve after the end, distinguishes different plant species by the welding curve of product.
Pcr amplification primer annealing temperature is between 50 ℃-60 ℃, and PCR product length is controlled between 40bp-100bp.Avoid comprising SNP site and In/del in the pcr amplification primer, PCR product sequence has tangible Tm value difference different between different target species to be identified as far as possible.The pcr amplification program is: 95 ℃ (pre-sex change 10min), 95 ℃ (sex change 30sec), Tm (renaturation 30sec), 72 ℃ of (extending 30sec) sex change, renaturation, three steps of extension circulate altogether and extend 10min, 16 ℃ (cooling) end after 72 ℃ after 55 times.What be used for that species identify can freely select canonical sequence at the different plant species optimum as required according to sequence, can be that COI gene order or other meet the sequence of biological label characteristics.
Embodiment 1
The evaluation of the nearly edge species of oyster during huge oyster belongs to:
Oyster species during huge oyster belongs to are many, and the oyster profile to be subjected to the influence of environment very big, be difficult to correctly to distinguish different plant species by phenotype.In species classification and evaluation, once once there were many disputes.Nowadays, trend towards adopting the differences of biological sequence label between different plant species such as mitochondrial COI gene sequence to identify different species gradually.
A. the acquisition of species appraisal basis sequence: belong to the interior different closely COI gene orders of edge oyster species with the domestic more common huge oyster of China, as species appraisal basis sequence.
B. design of primers: the sequence area of in above-mentioned oyster thing COI gene, selecting obviously to distinguish target species, utilize Primer Premier 5 softwares in this zone, to design the pcr amplification primer, primer extension product should have obvious Tm value difference different between different plant species, mainly is presented as G, C content difference.
Primer sequence is: COI-F1:TACTTAATATTGGGTTTTTAGGGT;
COI-R1：CGCGTATCAATATCCATTCC。
The c.PCR amplification: the genomic dna with oyster sample to be identified is template, utilizes above-mentioned primer to carry out pcr amplification.
The pcr amplification program is according to 95 ℃ (pre-sex change 10min), and 95 ℃ (sex change 30sec), Tm (renaturation 30sec), 72 ℃ of (extending 30sec) sex change, renaturation, three steps of extension circulate altogether and extend 10min, 16 ℃ (cooling) end after 72 ℃ after 55 times.
D. product detects and handles: after above-mentioned pcr amplification product was determined that with 1% agarose gel electrophoresis detection (Fig. 2) amplified production is single, 1 μ l LC-green was added in each reaction, is cooled to room temperature behind 95 ℃ of sex change 10min of regular-PCR instrument operation.
E. high resolving power melting curve (HRM) is analyzed: above-mentioned product moves HRM at the Lightscanner96 platform, collects the fluorescent signal data between 55 ℃-95 ℃.Utilize the analysis software that carries on the platform that fluorescent signal is changed into melting curve peak figure after the end; Distinguish different huge oysters according to the difference of the Tm value of the peak value correspondence of different curves and belong to nearly edge species (referring to Fig. 1).
5 kinds of huge oysters that present embodiment utilizes four coastal regions of China (Xiamen, Fujian, Nantong, Qingdao Tian Heng, Jiangnan, Qingdao) tideland to gather belong to oyster as the species expert evidence.Be the sequence of biological label characteristics with the COI gene, it is to identify that species information is clear and definite because these oyster samples carry out species through the method for traditional morphology, zoogeography and the comparison of COI gene sequencing.
As seen from Figure 1: melting curve gathers into 5 bundles respectively, presents 5 kinds of different Tm values respectively, and the huge oyster of 5 kinds of different plant species belongs to oyster in the counter sample.Type A is Crassostrea hongkongensis; Type B is Crassostrea ariakensis; Type C is Crassostrea angulata; Type D is Crassostrea sikamea; Type E is Crassostrea gigas.Above-mentioned differentiation result and sequencing sequence comparison result compare in full accord, and accuracy is 100%.
Claims (2)
1. the method identified of the nearly edge species of a seed oyster, it is characterized in that: be template with the oyster genomic dna, sequence label design primer according to each species gene group of oyster carries out pcr amplification, utilize amplified production at high resolving power melting curve (High Resolution Melting, HRM) the different and then differentiation oyster species of the Tm value difference in;
Be template with oyster genomic dna to be identified, carry out pcr amplification according to the COI gene order design primer of oyster, utilize the Tm value difference of amplified production in high resolving power melting curve (HRM) different and then distinguish the close species of shellfish; The sequence amplification primer is:
COI-F1：TACTTAATATTGGGTTTTTAGGGT；
COI-R1：CGCGTATCAATATCCATTCC。
2. by the method for the nearly edge species evaluation of the described oyster of claim 1, it is characterized in that: by the PCR product of above-mentioned amplification acquisition 76bp, denaturing treatment behind the interpolation nucleic acid saturable dye is cooled to room temperature and carries out HRM mensuration.
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| CN 201210189381 CN102758009B (en) | 2012-06-08 | 2012-06-08 | Method for identifying related species of oyster |
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| CN 201210189381 CN102758009B (en) | 2012-06-08 | 2012-06-08 | Method for identifying related species of oyster |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014125054A1 (en) * | 2013-02-15 | 2014-08-21 | Swiss Gemmological Institute Ssef | Dna profiling of pearls |
| CN104046683B (en) * | 2013-03-13 | 2015-06-10 | 中国科学院海洋研究所 | Method for discriminating two closely-related species of shellfish or identifying their hybrid generation |
| CN103605913B (en) * | 2013-12-05 | 2017-04-12 | 中国水产科学研究院黄海水产研究所 | Method applied to identification of pacific oyster family |
| CN105734134B (en) * | 2016-03-22 | 2019-03-26 | 中国水产科学研究院东海水产研究所 | A kind of method of molecular identificalion Crassostrea rivularis and Kumamoto oyster |
| CN107475414B (en) * | 2017-09-20 | 2021-06-08 | 中国科学院海洋研究所 | Method for screening parent oysters with high glycogen content |
| CN107354234B (en) * | 2017-09-20 | 2020-12-25 | 中国科学院海洋研究所 | Method for screening parent oysters with high glycogen content and related primer pair thereof |
| CN107557484A (en) * | 2017-10-30 | 2018-01-09 | 中国水产科学研究院东海水产研究所 | A kind of Kumamoto oyster based on DNA specific fragments（Crassostreasikamea）Authentication method |
| CN107723345A (en) * | 2017-11-10 | 2018-02-23 | 中国水产科学研究院东海水产研究所 | Crassostrea rivularis discrimination method based on DNA specific fragments |
| CN108642185A (en) * | 2018-03-29 | 2018-10-12 | 宁波大学 | A kind of molecular labeling and application for tiger spot cuttlefish with quasi- mesh cuttlefish species identification |
| CN108660218A (en) * | 2018-03-29 | 2018-10-16 | 宁波大学 | Molecular labeling for being identified between Mans needleless, tiger spot and quasi- mesh cuttlefish and application |
| CN108611422A (en) * | 2018-03-29 | 2018-10-02 | 宁波大学 | The molecular labeling with swordtip squid species identification and application for tiger spot, quasi- mesh |
| CN108642186A (en) * | 2018-03-29 | 2018-10-12 | 宁波大学 | Molecular labeling for being identified between Mans needleless, tiger spot and swordtip squid and application |
| CN108950007A (en) * | 2018-06-26 | 2018-12-07 | 中国计量大学 | For identifying the HRM primer and method of river Puffer and other fish products |
| CN114457167B (en) * | 2022-01-18 | 2023-11-28 | 秦皇岛市食品药品检验中心 | Method for detecting bivalve shellfish source components by fluorescence quantitative PCR |
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