CN107119143A - A kind of catfish DNA bar code standard detection sequence and its application - Google Patents
A kind of catfish DNA bar code standard detection sequence and its application Download PDFInfo
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- CN107119143A CN107119143A CN201710497312.1A CN201710497312A CN107119143A CN 107119143 A CN107119143 A CN 107119143A CN 201710497312 A CN201710497312 A CN 201710497312A CN 107119143 A CN107119143 A CN 107119143A
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Abstract
The invention discloses a kind of DNA bar code standard detection sequence of catfish and its application, its sequence such as SEQ ID NO:Shown in 1, the catfish method for identifying molecules of the present invention realizes the reliable DNA molecular identification technology used to the difficulty that catfish and Silurus Soldatovi Meridionalis and the identification of other kind catfish are brought, overcome needs the technological deficiencies such as the professional very familiar to catfish form to complete Individual identification in the prior art, compared with traditional Morphological Identification method, catfish can be quickly and accurately identified.
Description
Technical field
The present invention relates to species identification technical field, more particularly to a kind of catfish DNA bar code standard detection sequence and its
Application in species identification.
Background technology
Catfish (scientific name：Silurus asotus) belong to Actinopterygii Cypriniformes Cyprinidae crucian carp category, it is the peculiar thing of China
Kind.The similar of catfish is almost distributed across the whole world, most species be live in or the fresh water in rivers and creeks etc. in, but Some Species give birth to
Live in ocean.At present, the species identification to catfish is limited only to Morphological Identification, and professional accreditation personnel are seldom.Due to
Upper Yangtze River dam, which is built, causes spawning habitat to be destroyed；In addition the invasion and water pollution of alien species cause the existence of catfish
Environment subject to severe risks of damage, so the population quantity of catfish increasingly declines.Therefore, the local endemic species of catfish are effectively identified
And species conservation is very urgent.
DNA bar code technology (DNA barcoding) refer to in genome a segment standard, short DNA fragmentation reflects
One Molecular Identification new technology of earnest kind, can fast and accurately carry out species identification.At present most common point in animal
Son mark is the partial sequence of Mitochondrial cytochrome c oxidase subunit I (cytochrome coxidase I, COI) gene.
DNA bar code is with a wide range of applications, and studies and leads in species identification, molecular evolution, population genetic, endangered species protection etc.
Domain has certain development potentiality.DNA bar code traditional discrimination method different from the past, to the professional skill of researcher simultaneously
Without high requirement, up to biologist, under to common biological fan, the technology can be all grasped in a short time.DNA bar code
Technology has broken away from the obstacle that traditional form authentication method relies on protracted experience, can be achieved fast and accurately to identify, is Molecular Identification
Innovation in methodology, simple to operate, efficiency high, using it is wide the features such as will undoubtedly change the development side in whole species identification field
To.With DNA bar code technology, it can be very good fast and effectively to identify catfish.
Species identification is always the vital basic steps of research on taxology or even nearly all biological field.Cause
This, accurately to species identification, is particularly to those rare species and the species with Protection significance, taxonomic identification work is just aobvious
It must be even more important.Since 2003, since Canadian scientist Hebert is proposed with COI genes as DNA bar code, increasingly
Many research shows that DNA bar code has efficient feasibility on species taxonomy.Substantial amounts of result of study show using COI genes as
The DNA bar code of mark accurately can carry out species identification to all kinds of animals, can be used as various animal bars from COI genes
The standard bar code in code data storehouse.
This method is proved each other with traditional taxology method, and can not only solve that sample in identification is incomplete can not be accurate
Really the problems such as identification, and be conducive to Rapid identification.Special fish database is had at present, including more than 1.5 ten thousand kinds of fishes
The bar code data of class.But many fish of China are due to its peculiar property, without enough related datas.Also the phase without catfish
Correlation gene sequence.
The content of the invention
It is an object of the invention to propose a kind of catfish DNA bar code standard detection sequence and its answering in species identification
With.Catfish DNA bar code standard detection sequence provided by the present invention is advantageously implemented the Molecular Identification of catfish, can effectively shorten
Qualification time.
For up to this purpose, the present invention uses following technical scheme：
A kind of catfish DNA bar code standard detection sequence, the bar code standards detection sequence is COI genes, and its sequence is such as
SEQ ID NO:(168bp) shown in 1：
TTGCCCACGCAGGAGCTTCTGTAGATTTAACAATCTTTTCATTACATCTCGCAGGGGTGTCCTCCATTCTTGGAGCC
ATTAATTTCATTACAACCATTATTAATATGAAACCCCCAGCTATTTCACAATACCAAACACCTTTATTTGTATGAGC
CGTACTAATTACAG。
The primer pair of the catfish DNA bar code standard detection sequence above-mentioned for expanding, the forward primer of the primer pair
Sequence such as SEQ ID NO:Shown in 2, reverse primer is to sequence such as SEQ ID NO:Shown in 3.
Kit of the above-mentioned catfish DNA bar code standard detection sequence, above-mentioned primer in preparation for identifying catfish
In application.
A kind of kit for being used to identify catfish, it includes above-mentioned primer pair.The kit is also conventional including round pcr
Reagent.
A kind of recombinant vector, it includes above-mentioned catfish DNA bar code standard detection sequence.
Application of the above-mentioned catfish DNA bar code standard detection sequence, primer pair, kit in identification catfish.
A kind of method for identifying molecules of catfish, comprises the following steps：
(1) the separation and Extraction genomic DNA from tissue samples to be identified；
(2) as template, using pair of primers, expanded using genomic DNA obtained by step (1) by PCR
Increase；
The forward primer sequence and reverse primer sequences of the primer are respectively such as SEQ ID NO:2 and SEQ ID NO:3 institutes
Show；
SEQ ID NO:2 is as follows：
5’-TTGCCCACGCAGGAGCT-3’；
SEQ ID NO:3 is as follows：
5’-CTGTAATTAGTACGGCTCAT-3’；
(3) PCR amplification product obtained by step (2) is separated with agarose gel electrophoresis, according to expansion
Increase production the size result of determination of thing, if energy specific amplification goes out 170 ± 10bp band, carried out sequencing analysis；
(4) according to sequencing result, if with such as SEQ ID NO:The homology of nucleotide sequence shown in 1 98% with
On, you can judge that this to be identified is organized as catfish.
As a kind of optimal technical scheme, the condition of PCR is described in step (2)：95 DEG C of denaturation
5min；94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min, 40 circulations；72 DEG C of extension 10min.
The reaction system (25 μ L) of PCR described in step (2) is：10 × PCR Buffer2.5 μ L,
DNTPS 0.5 μ L, Taq (5U/ μ l) 0.2 μ L, MgCl2μ L, the NYCOI-R1 primer (10 of 1.5 μ L, NYCOI-F1 primer (10 μM) 1
μM) 1 μ L, DNA profiling (about 10ng/ μ l) 1 μ L, ddH2O supplies 25 μ L.
The method for identifying molecules flow chart of above-mentioned catfish is as shown in Figure 1.
Relative to prior art, the present invention has the advantages that：
(1) the invention provides a kind of DNA bar code standard detection sequence for being used to identify catfish.The DNA bar code mark
The application of quasi- detection sequence, realize to less catfish can not precise Identification individual and the close individual of form using can
The DNA molecular identification technology leaned on, fast and accurately identifies catfish；Overcome following deficiency of the prior art：To complete
Body identification needs the professional very familiar to catfish form；It is effective to shorten compared with traditional Morphological Identification method
Qualification time.
(2) the PCR reaction systems and reaction condition in the method for identifying molecules that the present invention is provided, repeatability is high, through multiple
Test stability good.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of the method for identifying molecules for the catfish that the present invention is provided.
Fig. 2 is the COI gene PCR amplification electroresis appraisal figures of the catfish musculature in embodiment 1；
Wherein, swimming lane M is DNA standard molecular weight marks, and swimming lane 1,2 is blank control, and swimming lane 3,4 is purpose fragment catfish flesh
The COI genes of meat tissue, the band that detection size is 170bp or so, swimming lane 5,6 is negative control.
Embodiment
Technical scheme is further illustrated below by embodiment.
Embodiment 1：
1st, collection, preservation and the processing of sample to be tested
The part musculature of clip catfish from immersed specimen, is stored in 95% alcohol and is deposited in -20 DEG C of refrigerators.
2nd, DNA is extracted
Biotech firm's kit method extracts the DNA in sample, is saved backup in -20 DEG C.
3rd, PCR primer is synthesized
This method chooses 1 pair of primer：
NYCOI-F1:5’-TTGCCCACGCAGGAGCT-3’(SEQ ID NO:2)；
NYCOI-R1:5’-CTGTAATTAGTACGGCTCAT-3’(SEQ ID NO:3)；
4th, PCR reaction systems (25 μ L)
5th, PCR response procedures
After reaction terminates, PCR instrument is opened, the sample for completing amplification is taken out, in 4 DEG C of preservations
6th, PCR primer is detected
2 μ l PCR primers are taken, with 1.5% agarose gel electrophoresis (120V, 200mA, 25min), genegreen dyes
Color.Gel imaging system detect, electrophoresis pattern as shown in Fig. 2 detection size be about 170 ± 10bp band, can preliminary judgement
Sample to be tested is probably catfish.
7th, gene sequencing
The selection preferable PCR primer of effect is delivered to biotech firm and is sequenced after purification, and sequencing result shows its sequence such as SEQ
ID NO:Shown in 4, SEQ ID NO:4 sequences are as follows：
TTGCCCACGCAGGAGCTTCTGTAGATTTAACAATCTTTTCATTACATCTCGCAGGGGTGTCCTCCATTC
TTGGAGCCATTAATTTCATTACAACCATTATTAATATGAAACCCCCAGCTATTTCACAATACCAAACACCTTTATTT
GTATGAGCCGTACTAATTACAG
Found through sequence analysis, SEQ ID NO:Nucleotide sequence shown in 4 and the nucleotides sequence as shown in SEQ ID NO.1
The homology of row is 99%, thus can determine that the sample to be tested is the tissue samples of catfish.
Embodiment 2：
2 groups of musculature samples are chosen from Fish Tissue sample (incomplete, it is impossible to carry out Biology identification), are stored in
In 95% alcohol.The musculature of selection adheres to the fish of two groups of different cultivars separately, and one of which is catfish (10 samples), another
Group is other kinds (10 samples), and sampling people carries out kind according to specifying information and records and number, and experiment operator is not
Tested in the case of clear 2 groups of sample kinds ownership.Tested using experimental method same as Example 1, PCR productions
Thing sequencing result shows that 10 samples of one of which are the tissue samples of catfish, and another group of 10 samples are the group of non-catfish
Sample is knitted, experimental result is completely the same with sampling people's record information.
Above-described embodiment is better embodiment of the invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Zhenjiang Hua Da detection Co., Ltd
<120>A kind of catfish DNA bar code standard detection sequence and its application
<130> 2017
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 168
<212> DNA
<213>Catfish Silurus asotus
<221>Catfish DNA bar code standard detection sequence
<400> 1
ttgcccacgc aggagcttct gtagatttaa caatcttttc attacatctc gcaggggtgt 60
cctccattct tggagccatt aatttcatta caaccattat taatatgaaa cccccagcta 120
tttcacaata ccaaacacct ttatttgtat gagccgtact aattacag 168
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence
<221> NYCOI-F1
<400> 2
ttgcccacgc aggagct 17
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<221> NYCOI-R1
<400> 3
ctgtaattag tacggctcat 20
<210> 4
<211> 168
<212> DNA
<213>It is unknown
<221>Sample P CR amplified production sequences
<400> 4
ttgcccacgc aggagcttct gtagatttaa caatcttttc attacatctc gcaggggtgt 60
cctccattct tggagccatt aatttcatta caaccattat taatatgaaa cccccagcta 120
tttcacaata ccaaacacct ttatttgtat gagccgtact aattacag 168
Claims (10)
1. a kind of catfish DNA bar code standard detection sequence, it is characterised in that its sequence such as SEQ ID NO:Shown in 1.
2. catfish DNA bar code standard detection sequence according to claim 1, it is characterised in that it is COI genes.
3. the primer pair for expanding the catfish DNA bar code standard detection sequence described in claim 1, it is characterised in that institute
State the forward primer sequence such as SEQ ID NO of primer pair:Shown in 2, reverse primer is to sequence such as SEQ ID NO:Shown in 3.
4. the primer described in catfish DNA bar code standard detection sequence, claim 3 described in claim 1 or 2 is preparing use
Application in the kit of identification catfish.
5. a kind of kit for being used to identify catfish, it is characterised in that it includes the primer pair described in claim 3.
6. kit according to claim 5, it is characterised in that the kit also includes round pcr common agents.
7. a kind of recombinant vector, it is characterised in that：Include the catfish DNA bar code standard detection sequence described in claim 1 or 2
Row.
8. primer pair, power described in catfish DNA bar code standard detection sequence as claimed in claim 1 or 2, claim 3
Profit requires application of the kit in identification catfish described in 4 or 5.
9. a kind of method for identifying molecules of catfish, it is characterised in that comprise the following steps：
(1) the separation and Extraction genomic DNA from tissue samples to be identified；
(2) as template, using pair of primers, expanded using genomic DNA obtained by step (1) by PCR；
The forward primer sequence and reverse primer sequences of the primer are respectively such as SEQ ID NO:2 and SEQ ID NO:Shown in 3；
(3) PCR amplification product obtained by step (2) is separated with agarose gel electrophoresis, produced according to amplification
The size result of determination of thing, if energy specific amplification goes out 170 ± 10bp band, is carried out sequencing analysis；
(4) according to sequencing result, if with such as SEQ ID NO:The homology of nucleotide sequence shown in 1 is more than 98%, i.e.,
It can determine whether that this to be identified is organized as catfish.
10. the method for identifying molecules of catfish according to claim 9, it is characterised in that polymerase chain described in step (2)
Formula reaction condition be：95 DEG C of denaturation 5min；94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min, 40 circulations；72 DEG C of extensions
10min。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107841564A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity primer and method of the Changjiang river original inhabitants catfish |
| CN107841563A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity labeled primers and method of catfish |
| CN108441502A (en) * | 2017-11-28 | 2018-08-24 | 江汉大学 | A kind of colter loach DNA bar code sequence and its application |
| CN109055400A (en) * | 2018-07-25 | 2018-12-21 | 江汉大学 | A kind of Usu intend Chang DNA bar code sequence and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107841564A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity primer and method of the Changjiang river original inhabitants catfish |
| CN107841563A (en) * | 2017-11-22 | 2018-03-27 | 镇江华大检测有限公司 | Differentiate the molecular specificity labeled primers and method of catfish |
| CN108441502A (en) * | 2017-11-28 | 2018-08-24 | 江汉大学 | A kind of colter loach DNA bar code sequence and its application |
| CN109055400A (en) * | 2018-07-25 | 2018-12-21 | 江汉大学 | A kind of Usu intend Chang DNA bar code sequence and its application |
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Application publication date: 20170901 |