CN107841563A - Differentiate the molecular specificity labeled primers and method of catfish - Google Patents
Differentiate the molecular specificity labeled primers and method of catfish Download PDFInfo
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- CN107841563A CN107841563A CN201711173774.4A CN201711173774A CN107841563A CN 107841563 A CN107841563 A CN 107841563A CN 201711173774 A CN201711173774 A CN 201711173774A CN 107841563 A CN107841563 A CN 107841563A
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Abstract
The present invention discloses a kind of molecular specificity primer and method for differentiating catfish.This is used for the sense primer NYF for differentiating the molecular specificity primer of catfish：As shown in SEQ ID NO.1：Anti-sense primer NYR：As shown in SEQ ID NO.2.The present invention utilizes molecular specificity labeled primers NYF/NYR, and quick Molecular Identification is carried out to catfish by conventional PCR method, and method is simple, is detected using round pcr, and the time is short, can complete within 1st, is effective auxiliary that morphological feature distinguishes catfish.
Description
Technical field
The present invention relates to a kind of molecular specificity labeled primers for differentiating catfish, and one kind to utilize the special primer to Nian
The method of fish sample quick discriminating.
Background technology
Catfish (scientific name：Silurus asotus) catfish, the animal kingdom-Chordata-subphylum chordata-spoke fin fish
Guiding principle-Silurformes-Nian section-Nian category-catfish kind.Catfish is similar to be almost distributed across the whole world, most species be live in pond or
In the fresh water in rivers and creeks etc., but Partial Species are lived in ocean.There is no squama on universal body, there is flat head and big mouth, mouth
Surrounding has several long hairs, must can identify taste using this, this is its feature.At present, the species identification of catfish is only limited to
In Morphological Identification, and professional accreditation personnel are seldom.Being built due to Upper Yangtze River dam causes spawning habitat to be destroyed；In addition
The invasion and water pollution of alien species cause the living environment subject to severe risks of damage of catfish, so the population quantity of catfish is increasingly
Decline.Therefore, it is very urgent that catfish place endemic species are carried out with effectively identification and species conservation.
DNA bar code technology (DNAbarcoding) refer to in genome a segment standard, short DNA fragmentation identifies
One Molecular Identification new technology of species, can fast and accurately carry out species identification.The most common molecule in animal at present
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, in research fields such as species identification, molecular evolution, population genetic, endangered species protections
With certain development potentiality.DNA bar code traditional discrimination method different from the past, has no to the professional skill of researcher
High requirement, up to biologist, under to common biological fan, can all grasp the technology in a short time.DNA bar code skill
Art 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 side
Jurisprudential innovation, 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, can be very good fast and effectively to identify catfish.
Species identification is always the vital basic steps of research on taxology or even almost all creatures field.Cause
This, accurately to species identification, the species particularly to those rare species and with Protection significance, taxonomic identification work just shows
It must be even more important.Since 2003, since Canadian scientist Hebert is proposed with COI genes as DNA bar code, increasingly
More 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 select COI genes as various animal bars
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 advantageous to Rapid identification.Existing special fish database at present, including more than 1.5 ten thousand kinds of fishes
The bar code data of class.But many fish in China are due to its peculiar property, without enough related datas.Also without the phase of catfish
Correlation gene sequence.
The content of the invention
It is an object of the invention in view of the shortcomings of the prior art, there is provided a kind of molecular specific for being used to differentiate catfish
Property labeled primer and its application.
Another object of the present invention is to provide a kind of method for differentiating catfish.
The purpose of the present invention can be achieved through the following technical solutions：
For differentiating the molecular specificity labeled primers of catfish, the nucleotide sequence of the primer is as follows：
Sense primer NYF:5’-TTGCCCACGCAGGAGCT-3’；
Anti-sense primer NYR:5’-CTGTAATTAGTACGGCTCAT-3’.
This is to use regular-PCR technology to molecular specificity primer, by the ribosomes ITS region sequences to catfish sample
Cloned sequence, then designed by NCBI sequence alignment, above-mentioned primer has high selectivity, and primer is carried out with this
PCR is expanded, and can obtain 170bp or so specific fragment.
The invention further relates to application of the above-mentioned molecular specificity labeled primers (NYF/NYR) in catfish is differentiated.
The invention further relates to above-mentioned molecular specificity labeled primers to prepare for differentiating answering in the kit of catfish
With.
It is a kind of to be used to differentiate the kit of catfish, include above-mentioned molecular specificity labeled primers in the kit.The examination
PCR common agents are also included in agent box.
A kind of method of quick discriminating catfish, using the genomic DNA of sample to be tested as template, with above-mentioned molecular specificity
Labeled primer (NYF/NYR) enters performing PCR amplification as amplimer, and electrophoresis detection is carried out to amplified production：If electrophoresis result
There are DNA purpose bands, then sample to be tested is catfish.Above-mentioned DNA purpose bands are 170 ± 10bp DNA purpose bands.
The response procedures of above-mentioned PCR amplification are：94 DEG C of denaturation 5min；94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min, 35
Circulation；72 DEG C of extension 10min.
Selection, extracting genome DNA, PCR reaction systems and the reaction condition expanded in the above method determines, and electrophoresis
Detection, these steps can be carried out according to this area conventional method.
It should be noted that the morphology that molecular specificity labeled primers of the present invention and detection method are only applicable to catfish is special
Preliminary judgement is levied, then will be deemed as the sample of catfish with the inventive method discriminating.
Comprised the following steps that to what discriminating catfish method of the present invention was verified：
(1) clip morphological feature is tentatively judged as the part musculature of catfish from immersed specimen, is stored in 95%
Deposited in alcohol in -20 DEG C of refrigerators.
(2) genomic DNA in biotech firm's kit method extraction sample, is saved backup in -20 DEG C.
(3) using the genomic DNA of step (2) extraction as template, made with the molecular specificity labeled primers (NYF/NYR)
For amplimer, enter performing PCR amplification：
PCR reaction systems (cumulative volume is 25 μ L) form：
PCR response procedures are：94 DEG C of pre-degeneration 5min；35 circulations (94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min)；72
Extend 10min under the conditions of DEG C.
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 figure 1, detection size be about 170 ± 10bp band, can preliminary judgement
Sample to be tested is probably catfish.
The beneficial effects are mainly as follows：
The molecular specificity labeled primers (NYF/NYR) of the present invention can carry out quick Molecular Identification, method letter to catfish
Single, accurate, high sensitivity, it is effective supplementary means that morphological feature distinguishes catfish.
Brief description of the drawings
Fig. 1 is the electrophoretogram for entering performing PCR amplification to catfish using the molecular specificity labeled primers of the present invention.
Wherein, M：DNA standard molecular weight marks；1,2 be blank；3,4 be purpose fragment；5,6 be negative control.
Embodiment
The present invention can be more objective and accurate from molecular level discriminating catfish.With reference to specific embodiment to this hair
It is bright to be further described.
Embodiment 1
(1) extraction of catfish genomic DNA to be measured
Catfish tissue sample is taken, is extracted after shredding using genome DNA extracting reagent kit, utilizes 1.5% agarose
Glue carries out electrophoresis detection to gained DNA, and uses UV spectrophotometer measuring DNA concentration, is deposited in -20 DEG C of refrigerators standby.
(2) design synthesis specific PCR amplimer
Specific primer of the design synthesis based on catfish, sense primer NYF：5 '-TTGCCCACGCAGGAGCT-3 ' and under
Swim primer NYR：5 '-CTGTAATTAGTACGGCTCAT-3 ', designed by technical staff, and synthesized by design of primers company.
(3) the PCR amplifications of molecular specificity labeled primers (NYF/NYR)
The μ L of PCR reaction systems 25：10 × PCR Buffer 2.5,0.5 0.2 μ L of μ L, Taq (5U/ μ l) of μ L, dNTPS,
MgCl2The μ L of 1.5 μ L, NYF primer (10 μM), 1 μ L, NYR primer (10 μM) 1, DNA profiling (about 10ng/ μ l) 1 μ L, ddH2O17.3μ
L。
PCR response procedures are：94 DEG C of pre-degeneration 5min；35 circulations (94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min)；72
Extend 10min under the conditions of DEG C.
(4) electrophoresis detection：
2 μ L PCR primers are taken, with 1.5% agarose gel electrophoresis (120V, 200mA, 25min), genegreen dyes
Color.Gel imaging system detects, and electrophoresis pattern is as shown in Figure 1.
M：DNA standard molecular weight marks；1,2 be blank；3,4 be purpose fragment；5,6 be negative control.As seen from Figure 1, expand
Increase the specific DNA band for that molecular weight is about 170bp or so.
Embodiment 2
Three kinds of catfish, Pelteobagrus fulvidraco, silver carp frost fish blocks are carried out with the method for embodiment 1 to detect, sampling people is according to specific letter
Breath carries out kind and records and number, and carries out experiment in the case of the unknown species name of experimenter, the results showed that only one
Kind of fish block it is amplifiable go out molecular weight be about 170bp or so specific DNA band, other two fish block sample can not amplify, examine
Survey result and sampling people's record information is completely the same.Therefore can be shown that the present invention nucleic acid molecule primers have it is high single-minded
Property, therefore can be used for quickly differentiating catfish.
Embodiment 3：
2 groups of musculature samples are chosen from Fish Tissue sample, are stored in 95% alcohol.The musculature of selection adheres to separately
The fish of two groups of different cultivars, one of which are catfish (5 samples), and another group is other kinds (5 samples), sample people's root
Kind is carried out according to specifying information to record and number, experiment operator is carried out in the case where not knowing 2 groups of sample kinds ownership
Experiment.Tested using experimental method same as Example 1, PCR primer sequencing result shows 5 samples of one of which
The specific DNA band that 170bp or so can be amplified is the tissue samples of catfish, and another group of 5 samples are non-catfish
Tissue samples, experimental result are 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 with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>Zhenjiang Hua Da detection Co., Ltd
<120>Differentiate the molecular specificity labeled primers and method of catfish
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ttgcccacgc aggagct 17
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctgtaattag tacggctcat 20
Claims (9)
1. the molecular specificity labeled primers for differentiating catfish, it is characterised in that：The nucleotide sequence of the primer is as follows：
Sense primer NYF:5’-TTGCCCACGCAGGAGCT-3’；
Anti-sense primer NYR:5’-CTGTAATTAGTACGGCTCAT-3’.
2. application of the molecular specificity labeled primers as claimed in claim 1 in catfish is differentiated.
3. application according to claim 2, it is characterised in that：, will with right using the genomic DNA of sample to be tested as template
Ask the molecular specificity labeled primers described in 1 to enter performing PCR amplification as amplimer, electrophoresis detection is carried out to amplified production：Such as
There are DNA purpose bands in fruit electrophoresis result, then sample to be tested is catfish.
4. molecular specificity labeled primers as claimed in claim 1 are preparing the application in being used to differentiate the kit of catfish.
5. a kind of kit for being used to differentiate catfish, it is characterised in that special comprising the molecule described in claim 1 in the kit
Different in nature labeled primer.
6. kit according to claim 5, it is characterised in that PCR common agents are also included in the kit.
A kind of 7. method of quick discriminating catfish, it is characterised in that：, will with right using the genomic DNA of sample to be tested as template
Ask the molecular specificity labeled primers described in 1 to enter performing PCR amplification as amplimer, electrophoresis detection is carried out to amplified production：Such as
There are DNA purpose bands in fruit electrophoresis result, then sample to be tested is catfish.
8. the method according to claim 4 for differentiating catfish, it is characterised in that：Described DNA purpose bands be 170 ±
10bp DNA purpose bands.
9. the method according to claim 4 for differentiating catfish, it is characterised in that：The response procedures of PCR amplification are：94
DEG C denaturation 5min；94 DEG C, 30s, 56 DEG C, 30s, 72 DEG C, 1min, 35 circulations；72 DEG C of extension 10min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711173774.4A CN107841563A (en) | 2017-11-22 | 2017-11-22 | Differentiate the molecular specificity labeled primers and method of catfish |
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| CN201711173774.4A CN107841563A (en) | 2017-11-22 | 2017-11-22 | Differentiate the molecular specificity labeled primers and method of catfish |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106256A (en) * | 2019-04-18 | 2019-08-09 | 中国水产科学研究院南海水产研究所 | A kind of molecular labeling primer and its application identifying egg-shaped pompano and Bu Shi silvery pomfret Scad |
| CN115323060A (en) * | 2021-06-11 | 2022-11-11 | 中国水产科学研究院珠江水产研究所 | Fish nuclear gene molecular marker primer, molecular marker and molecular marker database |
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| CN104073559A (en) * | 2014-06-25 | 2014-10-01 | 甘肃农业大学 | Identifying method for catfish based on DNA (deoxyribonucleic acid) Barcoding and raw meat products thereof and identifying kit |
| CN107058603A (en) * | 2017-06-27 | 2017-08-18 | 镇江华大检测有限公司 | A kind of catfish DNA bar code standard detection sequence and its application |
| CN107119143A (en) * | 2017-06-27 | 2017-09-01 | 镇江华大检测有限公司 | A kind of catfish DNA bar code standard detection sequence and its application |
-
2017
- 2017-11-22 CN CN201711173774.4A patent/CN107841563A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073559A (en) * | 2014-06-25 | 2014-10-01 | 甘肃农业大学 | Identifying method for catfish based on DNA (deoxyribonucleic acid) Barcoding and raw meat products thereof and identifying kit |
| CN107058603A (en) * | 2017-06-27 | 2017-08-18 | 镇江华大检测有限公司 | A kind of catfish DNA bar code standard detection sequence and its application |
| CN107119143A (en) * | 2017-06-27 | 2017-09-01 | 镇江华大检测有限公司 | A kind of catfish DNA bar code standard detection sequence and its application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110106256A (en) * | 2019-04-18 | 2019-08-09 | 中国水产科学研究院南海水产研究所 | A kind of molecular labeling primer and its application identifying egg-shaped pompano and Bu Shi silvery pomfret Scad |
| CN115323060A (en) * | 2021-06-11 | 2022-11-11 | 中国水产科学研究院珠江水产研究所 | Fish nuclear gene molecular marker primer, molecular marker and molecular marker database |
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