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 PDF

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CN105838802B
CN105838802B CN201610300523.7A CN201610300523A CN105838802B CN 105838802 B CN105838802 B CN 105838802B CN 201610300523 A CN201610300523 A CN 201610300523A CN 105838802 B CN105838802 B CN 105838802B
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threadfin
primer pair
squama
threadfins
primer
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CN105838802A (en
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蒙子宁
李活
杨翌聪
陈国清
庞德彬
林浩然
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MAOMING JINYANG TROPICAL FISH CULTIVATION Co Ltd
Sun Yat Sen University
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Sun Yat Sen University
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    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/686Polymerase chain reaction [PCR]
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/16Primer 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

The primer and method of molecular identificalion threadfin and more squama threadfins
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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CN112159851A (en) * 2020-10-28 2021-01-01 江苏省淡水水产研究所 Molecular identification method of eleutheronema tetradactylum and eleutheronema tetradactylum based on COI gene sequence
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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