CN105838802B - The primer and method of molecular identificalion threadfin and more squama threadfins - Google Patents
The primer and method of molecular identificalion threadfin and more squama threadfins Download PDFInfo
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- 241000791841 Alectis ciliaris Species 0.000 title claims abstract description 63
- 241001417968 Polynemidae Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000002773 nucleotide Substances 0.000 claims abstract description 17
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 17
- 238000012408 PCR amplification Methods 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000001962 electrophoresis Methods 0.000 claims description 8
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 claims description 6
- 230000004087 circulation Effects 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 239000012154 double-distilled water Substances 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims 1
- 239000002777 nucleoside Substances 0.000 claims 1
- 125000003835 nucleoside group Chemical group 0.000 claims 1
- 241000251468 Actinopterygii Species 0.000 description 7
- 230000000877 morphologic effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000003550 marker Substances 0.000 description 4
- 210000000006 pectoral fin Anatomy 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000000366 juvenile effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241001522631 Eleutheronema Species 0.000 description 1
- 241001224981 Eleutheronema rhadinum Species 0.000 description 1
- 241001522629 Eleutheronema tetradactylum Species 0.000 description 1
- 241000134214 Mugiliformes Species 0.000 description 1
- 241001502129 Mullus Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012850 discrimination method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 210000001614 vomer Anatomy 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- 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
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The invention discloses the primers of a kind of molecular identificalion threadfin and more squama threadfins, the primer includes primer pair 1 and primer pair 2, the primer pair 1 includes primers F 1 and R1, the primer pair 2 includes primers F 2 and R2, wherein the nucleotide sequence of primers F 1 such as SEQ ID NO:1 shows, the nucleotide sequence of primer R1 is as shown in SEQ ID NO:2, and the nucleotide sequence of primers F 2 is as shown in SEQ ID NO:3, and the nucleotide sequence of primer R2 is as shown in SEQ ID NO:4.The primer can identify threadfin and more squama threadfins on a molecular scale.The invention also discloses a kind of method of molecular identificalion threadfin and more squama threadfins, this method is efficient and convenient, accurate and reliable.
Description
Technical field
The invention belongs to molecular identificalions to be similar to nearly edge species technical field, and in particular to a kind of molecular identificalion threadfin and
The primer and method of more squama threadfins.
Background technique
Threadfin (Eleutheronema tetradactylum) and more squama threadfins (E.rhadinum) are mullet shapes
Two common species of mesh (Mugiliformes) Polynemidae (Polynemoidea) threadfin category (Eleutheronema), custom
Name noon fish, Ma You, fat content is high, fine and tender taste, protein abundance, and edible value with higher and economic value are
Rare high-end seawater fish.In China, the artificial breeding of threadfin and cultivation are still at the initial stage, and finished product fish, which relies primarily on, to be caught
Fishing, with increase in demand of the people to high-end rare marine product in recent years, the natural fishery resources of threadfin are just by overfishing
And failure increasingly, catch are difficult to meet the needs of market.
The many types formalness of Polynemidae is similar, diagnostic characteristics are not significant, is easy to obscure, main on morphological taxonomy
It is to be distinguished according to the free Filamentous fin ray number below pectoral fin, such as threadfin, six refer to threadfins, mostly finger threadfin.It belongs to
The threadfin and more squama threadfins that threadfin belongs to, pectoral fin lower section have 4 free Filamentous fin rays, remove pectoral fin color, side
Line squama number and vomer tooth plate are Bu Tong outer, other morphological features are almost the same, are often mistakenly thought of as same fish.Two kinds of fish
Not only closely similar in form, habitat is also similar, and often mixed to dwell in identical waters, the larva and juvenile of figure very little is just more difficult to recognize,
Very big difficulty is caused to the early life history and its supplement dynamic for further studying them, is unfavorable for protection and the pipe of resource
Reason.Therefore, a kind of method that is efficient and convenient, accurately and reliably identifying threadfin and more squama threadfins is needed.
Summary of the invention
The first purpose of the invention is to provide the primers of a kind of molecular identificalion threadfin and more squama threadfins, this draws
Object can identify threadfin and more squama threadfins on a molecular scale.
The object of the invention is also to provide a kind of method of molecular identificalion threadfin and more squama threadfins, this method
It is efficient and convenient, accurate and reliable.
The first purpose of this invention is achieved through the following technical solutions: a kind of molecular identificalion threadfin and more
The primer of squama threadfin, the primer include primer pair 1 and primer pair 2, and the primer pair 1 includes primers F 1 and R1, described
Primer pair 2 includes primers F 2 and R2, and wherein the nucleotide sequence of primers F 1 such as SEQ ID NO:1 shows, the nucleotides sequence of primer R1
Column are as shown in SEQ ID NO:2, and the nucleotide sequence of primers F 2 is as shown in SEQ ID NO:3, and the nucleotide sequence of primer R2 is such as
Shown in SEQ ID NO:4.
Specifically, each primer is as follows from 5 ' -3 ' nucleotide sequence:
F1:5 '-GCTCCTCCTCAGAGTTCCG-3 '；
R1:5 '-TCAATGATAATGCCCACCA-3 '；
F2:5 '-GGAAAGCGTGACATAGT-3 '；
R2:5 '-CAGTGAAGTCCAGGTAAT-3 '.
Second object of the present invention is achieved through the following technical solutions: a kind of molecular identificalion threadfin and more
The method of squama threadfin, comprising the following steps:
(1) above-mentioned primer pair 1 and primer pair 2 are designed；
(2) DNA of threadfin or more squama threadfins is extracted；
(3) using the DNA in step (2) as template, PCR amplification, amplified reaction knot are carried out using primer pair 1 and primer pair 2
Shu Hou, carries out electrophoresis detection, and electrophoresis result is shown in the vicinity 469bp and 606bp and is respectively provided with a specific DNA specificity
Band, sample detected is threadfin at this time, if there is not band in the vicinity 469bp or 606bp, for more squamas four finger
Threadfin.
In the method for above-mentioned molecular identificalion threadfin and more squama threadfins:
The DNA of threadfin or more squama threadfins is preferably extracted in step (2) using phenol chloroform organic solvent method.
Phenol chloroform organic solvent method is conventional phenol chloroform organic solvent method, and specific method can refer to document Sambroo J,
Fritsch E F, Maniatis T.MOLECULAR CLONING:A Laboratory Manual 2nd ed.Cold
Spring Harbor Laboratory Press, 1989.
When carrying out PCR amplification using primer pair 1 and primer pair 2 in step (3), preferably comprised in 20 μ LPCR reaction systems
2 10 μ L of 2 μ L of DNA, primer pair 1 or primer pair, premix Taq, dNTP, MgCl2PCR buffer 2 μ L, ddH2O 6μL。
When carrying out PCR amplification using primer pair 1 and primer pair in step (3), primer pair 1 and primer pair can be respectively adopted
2 carry out PCR amplification, can also carry out PCR amplification, if expanded with pair of primers, four using primer pair 1 and primer pair 2 simultaneously
Refer to that threadfin will appear a band 469bp (primers F 1 and R1) or 606bp (primers F 2 and R2), and more squama threadfins are not in
Band.If simultaneously with two pairs of primer amplifications, two band 469bp (primers F 1 and R1) and 606bp can occur simultaneously in threadfin
(primers F 2 and R2), and more squama threadfins are not in band.
When carrying out PCR amplification using primer pair 1 in step (3), the recommendation of PCR response procedures is: 95 DEG C of 5min first, then 94
DEG C 30s, 66 DEG C of 30s, 72 DEG C of 45 totally 32 circulation, then 72 DEG C of 10min；When carrying out PCR amplification using primer pair 2, PCR reaction interval
Sequence recommendation is: 95 DEG C of 5min first, then 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 45 totally 32 circulation, then 72 DEG C of 10min.
When carrying out electrophoresis detection in step (3), it is preferred to use the agarose gel electrophoresis that mass percentage is 1.5% is examined
It surveys, and is observed under ultraviolet light after Redsafe is added.
The present invention has the advantage that
(1) present invention devises the primer of molecular identificalion threadfin and more squama threadfins for the first time, which can divide
Identify threadfin and more squama threadfins in sub- level；
(2) the method for the present invention is that the one kind established for the first time both at home and abroad identifies threadfin on a molecular scale and more squamas four refer to
The new method of threadfin, it utilizes 4 specific primers, and threadfin and more squamas four can be identified by completing a PCR reaction
Refer to threadfin；
(3) it compares and morphology discrimination method, the method for the present invention is not necessarily to rely on the professional knowledge of taxonomy of fishes, more
Importantly, can not only identify threadfin and more squama threadfin finished product fishes, the larva and juvenile that can also identify them, fish-egg are very
To being marine product after processing, has the characteristics that simple and efficient, accurate stable, objective and fair, economical and practical.
Detailed description of the invention
Fig. 1 is using the key band of primers F 1 and the R1 threadfin expanded in embodiment 2, and wherein M is DNA
Marker (DL2000), S are threadfins, and D is more squama threadfins；
Fig. 2 is using the key band of primers F 2 and the R2 threadfin expanded in embodiment 2, and wherein M is DNA
Marker (DL2000), S are threadfins, and D is more squama threadfins；
Fig. 3 is using the electrophoresis detection of primers F 1 and the R1 threadfin expanded in embodiment 3 as a result, wherein M is DNA
Marker (DL2000), 1-4 are threadfin samples；
Fig. 4 is using the electrophoresis detection of primers F 2 and the R2 threadfin expanded in embodiment 3 as a result, wherein M is DNA
Marker (DL2000), d1-d4 are more squama threadfin samples.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.
Embodiment 1
The primer of molecular identificalion threadfin provided in this embodiment and more squama threadfins, primer include 1 He of primer pair
Primer pair 2, primer pair 1 include primers F 1 and R1, and primer pair 2 includes primers F 2 and R2, and wherein the nucleotide sequence of primers F 1 is such as
SEQ ID NO:1 shows that the nucleotide sequence of primer R1 is as shown in SEQ ID NO:2, the nucleotide sequence of primers F 2 such as SEQ ID
Shown in NO:3, the nucleotide sequence of primer R2 is as shown in SEQ ID NO:4.
The nucleotide sequence of each primer 5 ' -3 ' is specific as follows:
F1:5 '-GCTCCTCCTCAGAGTTCCG-3 '；
R1:5 '-TCAATGATAATGCCCACCA-3 '；
F2:5 '-GGAAAGCGTGACATAGT-3 '；
R2:5 '-CAGTGAAGTCCAGGTAAT-3 '.
Primer pair 1 and primer pair 2 in the present invention obtain in the following manner: first using the random primer of 10bp as primer
PCR amplification (PCR amplification condition can be with embodiment 2) is carried out to threadfin and more squama threadfins, it is random by filtering out
The specific fragment that primer amplification obtains, again one end 10~20bp sequence is sequenced plus original 10bp with power traction after cloning and sequencing
Object primers, PCR amplification filters out the primer that can go out specific band again (as shown in above).
Embodiment 2
After professional Morphological Identification, the multiple threadfins and more squama threadfins of known kind are chosen, using normal
After rule phenol chloroform organic solvent method extracts its DNA, using 4 kinds of primers, PCR amplification is carried out to its genome.
It include 2 μ L of DNA, 10 μ L of primer (F1/R1, F2/R2) in 20 μ LPCR reaction systems；Premix Taq, dNTP, MgCl2
PCR buffer 2 μ L, ddH2O 6μL。
PCR response procedures are 95 DEG C of 5min, [94 DEG C of 30sec → 66 DEG C (primers F 1, R1) or 56 DEG C (primers F 2, R2)
30sec → 72 DEG C 45sec] × → 72 DEG C of 10min of 32 circulations.
PCR reaction terminates, and takes 5 μ L products to be detected with 1.5% agarose gel electrophoresis, is added after Redsafe ultraviolet
It is observed under light.
As a result, it has been found that it is 469bp (primers F 1 and R1) and 606bp that a size occur simultaneously in multiple threadfin samples
The specific DNA band of (primers F 2 and R2), specifically as shown in figs. 1-2.
It is 469bp (primers F 1 and R1) and 606bp (primers F 2 that opposite multiple more squama threadfin samples, which do not occur size,
And R2) specific DNA band illustrate as shown in figs. 1-2 when distinguishing PCR amplification using primer pair 1 and primer pair 2, when
It is threadfin sample, instead when 469bp (primers F 1 and R1) and 606bp (primers F 2 and R2) specific band occurs simultaneously
Then be more squama threadfin samples.
Embodiment 3
The method of molecular identificalion threadfin provided in this embodiment and more squama threadfins, comprising the following steps:
(1) above-mentioned primer pair 1 and primer pair 2 are designed, detailed process is refering to embodiment 1；
(2) threadfin or more squama threadfins for respectively selecting the mature individual of 8 tails, using conventional phenol chloroform organic solvent method
Extract the DNA of threadfin or more squama threadfins；
(3) using the DNA in step (2) as template, PCR amplification is carried out using primer pair 1 and primer pair 2 respectively.
It include 2 μ L of DNA, 10 μ L of primer (F1/R1, F2/R2) in 20 μ LPCR reaction systems；Premix Taq, dNTP, MgCl2
PCR buffer (buffer) 2 μ L, ddH2O 6μL。
PCR response procedures are 95 DEG C of 5min, [94 DEG C of 30sec → 66 DEG C (primers F 1, R1) or 56 DEG C (primers F 2, R2)
30sec → 72 DEG C 45sec] × → 72 DEG C of 10min of 32 circulations.
PCR reaction terminates, and takes the agarose gel electrophoresis that 5 μ L product mass percentages are 1.5% to detect, is added
It is observed under ultraviolet light after Redsafe.
As a result, it has been found that 4 parts of Morphological Identifications are the sample of threadfin while one size of appearance is 469bp (primers F 1
And R1) or 606bp (primers F 2 and R2) specific DNA band, show sample be threadfin, as shown in Figure 3-4.
4 parts of Morphological Identifications are that the sample of more squama threadfins band does not occur, show that sample is more squama threadfins, this
One result and the result of Morphological Identification are completely the same, as shown in Figure 3-4.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included in protection scope of the present invention.
Claims (6)
1. the primer of a kind of molecular identificalion threadfin and more squama threadfins, it is characterized in that: the primer includes 1 He of primer pair
Primer pair 2, the primer pair 1 include primers F 1 and R1, and the primer pair 2 includes primers F 2 and R2, wherein the nucleosides of primers F 1
Acid sequence is as shown in SEQ ID NO:1, and the nucleotide sequence of primer R1 is as shown in SEQ ID NO:2, the nucleotides sequence of primers F 2
Column are as shown in SEQ ID NO:3, and the nucleotide sequence of primer R2 is as shown in SEQ ID NO:4.
2. a kind of method of molecular identificalion threadfin and more squama threadfins, it is characterized in that the following steps are included:
(1) primer pair 1 and primer pair 2 in claim 1 are designed；
(2) DNA of threadfin or more squama threadfins is extracted；
(3) using the DNA in step (2) as template, PCR amplification is carried out using primer pair 1 and primer pair 2, after amplified reaction,
Electrophoresis detection is carried out, electrophoresis result is shown in the vicinity 469bp and 606bp and is respectively provided with a specific DNA specific band,
Sample detected is threadfin at this time, if there is not band in the vicinity 469bp or 606bp, for more squama threadfins.
3. the method for molecular identificalion threadfin according to claim 2 and more squama threadfins, it is characterized in that: step
(2) DNA of threadfin or more squama threadfins is extracted in using phenol chloroform organic solvent method.
4. the method for molecular identificalion threadfin according to claim 2 and more squama threadfins, it is characterized in that: step
(3) when carrying out PCR amplification using primer pair 1 and primer pair 2 in, in 20 μ LPCR reaction systems comprising 2 μ L of DNA, primer pair 1 or
2 10 μ L of primer pair, premix Taq, dNTP, MgCl2PCR buffer 2 μ L, ddH2O 6μL。
5. the method for molecular identificalion threadfin according to claim 2 and more squama threadfins, it is characterized in that: step
(3) when carrying out PCR amplification using primer pair 1 in, PCR response procedures are: 95 DEG C of 5min first, then 94 DEG C of 30s, 66 DEG C of 30s, 72
DEG C 45 totally 32 circulations, then 72 DEG C of 10min；When carrying out PCR amplification using primer pair 2,95 DEG C of 5min first, then 94 DEG C of 30s, 56
DEG C 30s, 72 DEG C of 45 totally 32 circulation, then 72 DEG C of 10min.
6. the method for molecular identificalion threadfin according to claim 2 and more squama threadfins, it is characterized in that: step
(3) when carrying out electrophoresis detection in, mass percentage is used to detect for 1.5% agarose gel electrophoresis, and Redsafe is added
It observes under ultraviolet light afterwards.
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| CN116144788B (en) * | 2022-10-21 | 2024-03-01 | 中山大学 | SSR (simple sequence repeat) marker primer, method and application for evaluating genetic diversity of eleutheronema tetradactylum population |
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| 基于 cox1条形码的鱼肚DNA分子鉴定;朱惠敏 等;《广东海洋大学学报》;20141231;第34卷(第6期);第1-5页 |
| 基于COI基因序列的中国东南沿海四指马鲅属鱼类的遗传多样性分析;邓春兴;《中国优秀硕士学位论文全文数据库 农业科技辑》;20150315(第03期);第D052-83页 |
| 多鳞四指马鱼友不同地理群体的形态差异及遗传变异研究;杨阳;《中国优秀硕士学位论文全文数据库 农业科技辑》;20140515(第05期);第D052-28页 |
| 应用AFLP技术分析多鳞四指马鲅不同地理群体的遗传多样性;杨阳 等;《海洋渔业》;20130531;第35卷(第2期);第131-136页 |
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