CN109097481A - Method based on high throughput sequencing technologies identification fish-egg larva and juvenile - Google Patents
Method based on high throughput sequencing technologies identification fish-egg larva and juvenile Download PDFInfo
- Publication number
- CN109097481A CN109097481A CN201810954202.8A CN201810954202A CN109097481A CN 109097481 A CN109097481 A CN 109097481A CN 201810954202 A CN201810954202 A CN 201810954202A CN 109097481 A CN109097481 A CN 109097481A
- Authority
- CN
- China
- Prior art keywords
- juvenile
- fish
- sequence
- high throughput
- larva
- 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
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
- C12Q1/6869—Methods for sequencing
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the methods based on high throughput sequencing technologies identification fish-egg larva and juvenile, and field acquisition fish-egg larva and juvenile sample, Locale Holding is in 95% alcohol；Sample gene group DNA is extracted using DNeasy Blood&Tissue Kit kit；PCR amplification is carried out to said gene group DNA using Cytb universal primer；High-flux sequence is carried out to pcr amplification product；The gene order of acquisition is subjected to sequence screening, is compared, it is compared by the sequence and sequence in public database NCBI that are generated through high-throughput gene sequencing platform, judge that fish-egg larva and juvenile species belong to according to gene order similarity index, entire method process is not required to each sample extraction DNA, PCR amplification and sequencing, the single species identification taken time and effort compared to tradition, high throughput method can carry out species identification to a large amount of complex samples in a short time, time saving and labor saving.
Description
Technical field
The present invention relates to fishery resources field of biology, are based especially on high throughput sequencing technologies identification fish-egg larva and juvenile
Method
Background technique
Fish early stage individual (including fish-egg, larva and juvenile) are the important sources of fish population supplement, Species distributing sum number
Amount variation is the most direct effective data information of assessment sea area fish spawning ground, parent population stock number, the amount of marketable aquatic animals which can be caught to supplement fishery resources.Tradition
The method of identification fish early stage individual is mainly based on Morphological Identification, but metamorphosis is multiple during fish early development
It is miscellaneous, and morphological feature interspecific difference is not significant, taxonomic identification work difficulty is larger, and traditional form method excessively relies on mirror
The expert's conclusion of the personal experience for the person of determining and Observations Means, different assessors is often inconsistent, or even occurs accidentally reflecting.
Molecular biology method is emerging fish early stage Individual identification method, and this method utilizes the stability of inhereditary material
It is identified for early development stage individual, the limitation of identification by morphological characters species can only be relied on by breaching traditional taxonomy,
Precise Identification for fish early stage individual provides new method.Currently, being mainly base using more molecular biology method
In the PCR method of sequencing, carry out sequencing after segment obtained by PCR amplification is purified, and with the resulting expansion of same primer
Increase fragment sequence to be compared and complete species identification.Conventional gene order detection technique, usually can only once detect one
Kind species, and the gene order of a large amount of mixed species cannot be quickly analyzed, and in most cases, the fish early stage of field acquisition
Body sample is usually a large amount of, the mixture of a variety of different fish-eggs or larva and juvenile.
The high throughput sequencing technologies risen in recent years revolutionize the mode of DNA analysis, with Illumina microarray dataset
For, technical characteristics are exactly that random can be attached to be sequenced in synthesis, after DNA fragmentation adds connector
Glass surface (Flow cell) forms the DNA molecular cluster with cloned dna molecule segment by bridge-type PCR amplification；Based on can
The inverse principle for terminating chemical reaction carries out large-scale parallel sequencing to millions of a segments simultaneously；After sequencing reaction, imaging
System can capture the nucleotide of fluorescent marker, and the image data of one of cluster is exactly a DNA sequence dna；Then by picture number
Base sequence information is converted to according to it, completes sequencing.Unique DNA molecule can only be sequenced with traditional Sanger sequencing approach
Difference, the technology can obtain the sequence of each DNA molecular in sample, have many advantages, such as that flux is high, speed is fast, and can be simultaneously
Multiple species in detection of complex sample.
Summary of the invention
Technical problem to be solved by the present invention lies in provide the side based on high throughput sequencing technologies identification fish-egg larva and juvenile
Method has many advantages, such as that flux is high, speed is fast, and can by the way that high throughput sequencing technologies are applied in identification fish-egg larva and juvenile
With multiple species in detection of complex sample simultaneously.
In order to solve the above technical problems, the technical solution of the invention is as follows:
Based on the method for high throughput sequencing technologies identification fish-egg larva and juvenile, the described method comprises the following steps:
S1: field acquisition fish-egg larva and juvenile sample, Locale Holding is in 95% alcohol；
S2: sample gene group DNA is extracted using DNeasy Blood&Tissue Kit kit；
S3: PCR amplification is carried out to genomic DNA described in S2 using Cytb universal primer；
S4: high-flux sequence is carried out to pcr amplification product, obtains the sequence information of target gene；
S5: the gene order of acquisition is subjected to sequence screening, is compared, the sequence generated through high-throughput gene sequencing platform is passed through
Column are compared with sequence in public database NCBI, judge that fish-egg larva and juvenile species belong to according to gene order similitude.
Further, the sample to be tested in the S1 includes but is not limited to fresh biopsy tissue, fresh epidermal tissue.
Further, the reaction system of PCR amplification is 25 μ L in the S3, is specifically included: 2.5 10 × amplifications of μ L are slow
Fliud flushing, 2 μ L mole coefficients are the dNTPs of 2.5mmol/L, and 0.5 μ L mole coefficient is the positive primer of Cytb of 10 μm of ol/L, 0.5 μ L
Mole coefficient is the Cytb anti-primer of 10 μm of ol/L, and 0.3 μ L Taq DNA polymerase, 1 μ L of template DNA, remaining is mended with ultrapure water
Foot.
Further, in the S3 PCR amplification specific reaction condition are as follows: template DNA be heated to 94 DEG C carry out it is pre-
Denaturation, duration 4min carry out denaturation 30s at 94 DEG C；When template DNA it is heated denaturation at it is single-stranded after, temperature is down to 50
DEG C carry out annealing 30s；Then extension 1min is carried out at 72 DEG C, it is same to extend 35 circulations, 72 DEG C of extension 9min.
Further, platform used in high-flux sequence is carried out in the S4 includes but is not limited to 454FLX pyrophosphoric acid
Microarray dataset, Solexa genome analysis platform, SOLiD sequenator.
Further, high-flux sequence is carried out in the S4, comprising: the Taq DNA polymerase being transformed is added and has
The dNTPs of fluorescent marker detects the dNTPs signal in conjunction with DNA fragmentation to obtain base information, and circulation only synthesizes one every time
A base knows the sequence information of target gene by counting the fluorescence signal that every wheel is collected into.
Further, the gene order similarity index in the S5 specifically: genetic similarty >=99% is attributed to together
Kind；Genetic similarty 92%~99% is to belong to；Genetic similarty 85%~92% is equal.
After adopting the above scheme, high throughput sequencing technologies can be applied to the identification of fish-egg larva and juvenile by the present invention, can be with
More quickly and accurately type complicated included in the fish-egg of field acquisition, larva and juvenile sample is identified, to meet with
The taxonomic identification of the fish early stage individual of bottleneck provides a new research means.
Specific embodiment
The invention will be further described combined with specific embodiments below.It should be noted that disclosed below
Technical characteristic involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.
Therefore, the model of claimed invention is not intended to limit to the detailed description of the embodiment of the present invention below
It encloses, but is merely representative of selected embodiment of the invention.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
Every other embodiment obtained under the premise of creative work is made, shall fall within the protection scope of the present invention.
Embodiment
Using high throughput sequencing technologies identification fish-egg, specific step is as follows for larva and juvenile:
(1) acquire experiment sample: field acquisition fish-egg larva and juvenile sample, Locale Holding are taken back in 95% alcohol
Laboratory carries out lab analysis, and sample here includes but is not limited to fresh biopsy tissue, fresh epidermal tissue；
(2) it extracts sample genomic dna: extracting sample gene group using DNeasy Blood&Tissue Kit kit
DNA；
(3) PCR amplification: expanding sample gene group DNA using Cytb universal primer, and reaction system is 25 μ L, tool
Body includes 2.5 μ 10 × amplification buffers of L, and 2 μ L mole coefficients are the dNTPs of 2.5mmol/L, and 0.5 μ L mole coefficient is 10 μ
The positive primer of the Cytb of mol/L, 0.5 μ L mole coefficient are the Cytb anti-primer of 10 μm of ol/L, 0.3 μ L Taq DNA polymerase, mould
Plate DNA1 μ L, remaining is supplied with ultrapure water；
Specific reaction condition are as follows: template DNA is heated to 94 DEG C of progress initial denaturations, duration 4min, at 94 DEG C into
Row denaturation 30s；When template DNA it is heated denaturation at it is single-stranded after, temperature is down to 50 DEG C and carries out annealing 30s；Then it is carried out at 72 DEG C
Extend 1min, it is same to extend 35 circulations, 72 DEG C of extension 9min.
(4) high-flux sequence: high-flux sequence is carried out to pcr amplification product by high-flux sequence platform, transformation is added
The Taq DNA polymerase crossed and the dNTPs with fluorescent marker detect the dNTPs signal in conjunction with DNA fragmentation to obtain alkali
Base information, each circulation only synthesize a base, know the sequence of target gene by counting the fluorescence signal that every wheel is collected into
Column information.
In the present embodiment, microarray dataset includes but is not limited to 454FLX pyrosequencing platform, Solexa gene component
Analyse platform, SOLiD sequenator.
(5) biological information compares analysis: the gene order of acquisition is subjected to bioinformatic analysis, including sequence screening,
It compares, comparison is to compare the sequence that high-flux sequence platform generates with sequence in public database NCBI, passes through sequence
Similitude judges that fish-egg larva and juvenile species belong in field acquisition sample.
It is same by fish-egg larva and juvenile sample identification according to genetic similarty >=99%, 92%~99%, 85%~92%
Kind belongs to, the level of section.
Whole experiment process is not required to the list to each sample extraction DNA, PCR amplification and sequencing, taken time and effort compared to tradition
One species identification, high throughput method can carry out species identification to a large amount of complex samples in a short time, time saving and labor saving.
Xiamen sea area is successfully mixed fish-egg, larva and juvenile sample identification to 25 types by we on a small quantity, is under the jurisdiction of 7 mesh, 18 section
20 belong to, and still have 9 types only to identify category, 6 types only identify section, it was demonstrated that high throughput sequencing technologies are in fish, larva and juvenile
Applicability in Identification of Species is appropriate for wider fish-egg, larva and juvenile sample mirror by screening, optimization, design in next step
Fixed gene and primer improves the technical appraisement fish-egg, the range of larva and juvenile and precision.
The above described is only a preferred embodiment of the present invention, be not intended to limit the scope of the present invention,
Therefore the changes or modifications that claim under this invention and specification are done in every case, it all should belong to the range that the invention patent covers
Within.
Claims (7)
1. the method based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that the described method comprises the following steps:
S1: field acquisition fish-egg larva and juvenile sample, Locale Holding is in 95% alcohol；
S2: sample gene group DNA is extracted using DNeasy Blood&Tissue Kit kit；
S3: PCR amplification is carried out to genomic DNA described in S2 using Cytb universal primer；
S4: high-flux sequence is carried out to pcr amplification product, obtains the sequence information of target gene；
S5: carrying out sequence screening for the gene order of acquisition, compare, the sequence by being generated through high-throughput gene sequencing platform and
Sequence compares in public database NCBI, judges that fish-egg larva and juvenile species belong to according to gene order similarity index.
2. the method according to claim 1 based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that institute
Stating the sample to be tested in S1 includes but is not limited to fresh biopsy tissue, fresh epidermal tissue.
3. the method according to claim 1 based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that institute
The reaction system for stating PCR amplification in S3 is 25 μ L, and specifically include: 2.5 μ 10 × amplification buffers of L, 2 μ L mole coefficients are
The dNTPs of 2.5mmol/L, 0.5 μ L mole coefficient are the positive primer of Cytb of 10 μm of ol/L, and 0.5 μ L mole coefficient is 10 μm of ol/L
Cytb anti-primer, 0.3 μ L Taq DNA polymerase, 1 μ L of template DNA, remaining is supplied with ultrapure water.
4. the method according to claim 1 based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that institute
State the specific reaction condition of PCR amplification in S3 are as follows: template DNA is heated to 94 DEG C of progress initial denaturations, duration 4min,
Denaturation 30s is carried out at 94 DEG C；When template DNA it is heated denaturation at it is single-stranded after, temperature is down to 50 DEG C and carries out annealing 30s；Then exist
72 DEG C carry out extension 1min, same to extend 35 circulations, 72 DEG C of extension 9min.
5. the method according to claim 1 based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that institute
Stating and carrying out platform used in high-flux sequence in S4 includes but is not limited to 454FLX pyrosequencing platform, Solexa gene
Group analysis platform, SOLiD sequenator.
6. the method according to claim 1 based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that institute
It states and carries out high-flux sequence in S4, comprising: the Taq DNA polymerase being transformed and the dNTPs with fluorescent marker is added,
The dNTPs signal in conjunction with DNA fragmentation is detected to obtain base information, circulation only synthesizes a base every time, every by counting
The fluorescence signal that is collected into is taken turns to know the sequence information of target gene.
7. the method according to claim 1 based on high throughput sequencing technologies identification fish-egg larva and juvenile, which is characterized in that institute
State the gene order similarity index in S5 specifically: genetic similarty >=99% is attributed to of the same race；Genetic similarty 92%~
99% is to belong to；Genetic similarty 85%~92% is equal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810954202.8A CN109097481A (en) | 2018-08-21 | 2018-08-21 | Method based on high throughput sequencing technologies identification fish-egg larva and juvenile |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810954202.8A CN109097481A (en) | 2018-08-21 | 2018-08-21 | Method based on high throughput sequencing technologies identification fish-egg larva and juvenile |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109097481A true CN109097481A (en) | 2018-12-28 |
Family
ID=64850514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810954202.8A Pending CN109097481A (en) | 2018-08-21 | 2018-08-21 | Method based on high throughput sequencing technologies identification fish-egg larva and juvenile |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097481A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5786144A (en) * | 1996-05-13 | 1998-07-28 | American Museum Of Natural History | Method and compositions for identification of species origin of caviar |
| CN107557478A (en) * | 2017-09-15 | 2018-01-09 | 中国长江三峡集团公司中华鲟研究所 | A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer |
-
2018
- 2018-08-21 CN CN201810954202.8A patent/CN109097481A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5786144A (en) * | 1996-05-13 | 1998-07-28 | American Museum Of Natural History | Method and compositions for identification of species origin of caviar |
| CN107557478A (en) * | 2017-09-15 | 2018-01-09 | 中国长江三峡集团公司中华鲟研究所 | A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer |
Non-Patent Citations (4)
| Title |
|---|
| 徐春燕等: "基于COI基因的福建近海部分仔稚鱼DNA条形码分析", 《中国水产科学》 * |
| 李洋: "基于DNA条形码的长江上游江津段鱼类早期资源种类鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 胡大一等: "《运动性心律失常与猝死》", 31 July 2017, 河南科学技术出版社 * |
| 郑芳等: "《临床分子诊断学》", 31 July 2014, 华中科技大学出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200263168A1 (en) | High throughput transcriptome analysis | |
| RU2752700C2 (en) | Methods and compositions for dna profiling | |
| CN103384725A (en) | Fetal genetic variation detection | |
| CN109554488B (en) | Female sturgeon specific DNA fragment and application thereof | |
| CN105483123A (en) | Genetic marker combination, individual gene identity certificate and uses thereof | |
| KR20200047686A (en) | Normalization for sequencing library | |
| CN104017859A (en) | Method for identifying sugarcane germplasm resources based on SSR (Simple Sequence Repeats) and CE (capillary electrophoresis) technique | |
| Garosi et al. | Defining best practice for microarray analyses in nutrigenomic studies | |
| KR101353083B1 (en) | SNP markers and methods for highly fetile pig | |
| CN111549146A (en) | Universal macro-barcode amplification primer for mitochondria of amphibian and application method thereof | |
| WO2011146942A1 (en) | Methods and kits to analyze microrna by nucleic acid sequencing | |
| CN102321752B (en) | Fluorescence labeled detection kit for simultaneously analyzing 17 gene loci of canine genomic DNA, detection method and application thereof | |
| KR102194349B1 (en) | Single nucleotide polymorphism markers for parentage testing in Jeju black cattle and the uses thereof | |
| KR101843430B1 (en) | Generic marker for detecting korean cattle(Hanwoo) and chinese cattle(Yeonbyeonu) using Copy Number Variation(CNV) and method using the same | |
| US20220136043A1 (en) | Systems and methods for separating decoded arrays | |
| CN117144017B (en) | Molecular marker related to chicken growth traits and application thereof | |
| CN114875118B (en) | Methods, kits and devices for determining cell lineage | |
| CN106367498A (en) | Periplaneta americana microsatellite loci and application thereof | |
| CN106065417A (en) | A kind of STR classification system and kit | |
| CN108823327A (en) | Camphor tree full-length genome SSR molecular marker and its preparation method and application | |
| CN109097481A (en) | Method based on high throughput sequencing technologies identification fish-egg larva and juvenile | |
| CN109234412A (en) | The quickly method of the fast erythroculter ilishaeformis of the detection speed of growth and molecular labeling used | |
| CN106282332B (en) | Label and primer for multiple nucleic acid sequencing | |
| US10927405B2 (en) | Molecular tag attachment and transfer | |
| KR102017236B1 (en) | The primer set for analysis of cephalopods and methods for their qualitativeand quantitative analysis |
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 |
Application publication date: 20181228 |
|
| RJ01 | Rejection of invention patent application after publication |