CN106884053A - A kind of little yellow croaker and the PCR authentication methods and primer of large yellow croaker juvenile fish - Google Patents
A kind of little yellow croaker and the PCR authentication methods and primer of large yellow croaker juvenile fish Download PDFInfo
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- CN106884053A CN106884053A CN201710204070.2A CN201710204070A CN106884053A CN 106884053 A CN106884053 A CN 106884053A CN 201710204070 A CN201710204070 A CN 201710204070A CN 106884053 A CN106884053 A CN 106884053A
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- yellow croaker
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Abstract
The invention discloses a kind of little yellow croaker and the PCR authentication methods and primer of large yellow croaker juvenile fish, the PCR authentication methods step is：The genomic DNA of extraction sample, enters performing PCR and expands using the DNA for identifying primer pair extraction, and pcr amplification product is detected with agarose gel electrophoresis, and one amplified band of appearance is little yellow croaker, and two amplified bands of appearance are large yellow croaker.The present invention is simple and easy to do, and specificity is high, can clearly differentiate and distinguish form similar little yellow croaker and large yellow croaker juvenile fish.
Description
Technical field
The present invention relates to a kind of authentication method of species, the PCR identifications of more particularly to a kind of little yellow croaker and large yellow croaker juvenile fish
Method and primer.
Background technology
Little yellow croaker (Larimichthys polyactis) and large yellow croaker (Larimichthys crocea) are under the jurisdiction of perch
Shape mesh, Anabantoidei, Sciaenidae, yellow croaker category, four famous big marine products of Dou Zengshi China.Large yellow croaker is similar with little yellow croaker build,
Typically made a distinction with caudal peduncle ratios by the way that caudal peduncle is long, but for large yellow croaker and little yellow croaker juvenile fish, little yellow croaker and large yellow croaker exist
Juvenile fish stage form is very close, and due to small, measure characteristic carries out Identification of Species and there is larger error, and only from outside shape
It is very difficult that the two species are distinguished in state, is unfavorable for the protection of germ plasm resource.
For accurate evaluation rheum officinale fish resource, the rapid identification method for setting up a kind of little yellow croaker and large yellow croaker juvenile fish very must
Will.The discriminating of germ plasm resource separately has biochemistry and genetic fingerprints correlation technique in addition to traditional morphological taxonomy method.Wherein
Especially it is widely used with genetic fingerprint methods, sees the fields such as various criminal investigations, medical science, scientific research, is particularly applied in scientific research each
The difference of the similar species of class form and identification.
The content of the invention
It is simple and easy to do it is an object of the invention to a kind of little yellow croaker and the PCR authentication methods of large yellow croaker juvenile fish, specificity
Height, can clearly differentiate and distinguish form similar little yellow croaker and large yellow croaker juvenile fish.
Present invention also offers the primer for identifying little yellow croaker and large yellow croaker juvenile fish, high specificity.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of little yellow croaker and the PCR authentication methods of large yellow croaker juvenile fish, the PCR authentication methods step is：Extract sample
Genomic DNA, the DNA extracted using identification primer pair is entered performing PCR and expanded, and pcr amplification product is detected with agarose gel electrophoresis,
One amplified band of appearance is little yellow croaker, and two amplified bands of appearance are large yellow croaker；The identification primer sequence is as follows：
COI-F：5’-GTTACGGCACATGCYTTCGT-3’
COI-R1：5’-GGAAACACCTGCGAGGTGTA-3’
COI-R2：5’-TARAGGATGGGATCGCCTCC-3’.
The present invention uses molecular biology method, and the gene of little yellow croaker and large yellow croaker juvenile fish is extracted and screened, from
And make a distinction little yellow croaker and large yellow croaker juvenile fish the two different plant species, method is simple, and specificity is high.The present invention is used
Special primer amplification little yellow croaker and the DNA of large yellow croaker juvenile fish, then carry out gel electrophoresis analysis, can show both DNA
Work is distinguished, such that it is able to the similar little yellow croaker of form and large yellow croaker juvenile fish are also distinguished.
Little yellow croaker sample amplifies a band in 500bp positions, and each appearance at 300bp and 500bp of rheum officinale fish sample
One bright band, can be with quick discriminating little yellow croaker and large yellow croaker juvenile fish sample by banding pattern difference.
Preferably, the PCR amplification system is：10 × buffer 2.5 μ l, dNTP 2 μ l, COI-F 1 μ l, COI-R1
1 μ l, COI-R2 1 μ l, the μ l of Taq enzyme 0.15, DNA profiling 1 μ l, ddH2O supplies system to 25 μ l.
Preferably, final concentration of 1 μM of the final concentration of 0.8mM of dNTP, COI-F, final concentration of 1 μM of COI-R1,
Final concentration of 1 μM of COI-R2, the final concentration of 0.04U/ μ l of Taq enzyme.
Preferably, the PCR amplification conditions are：94 DEG C of predegeneration 3min, after by 40 circulations, each circulation includes
94 DEG C of 45s, 50 DEG C of 45s, 72 DEG C of 45s；Last 72 DEG C of extensions 10min.
A kind of primer for identifying little yellow croaker and large yellow croaker juvenile fish, including three specific primers, primer sequence are as follows：
COI-F：5’-GTTACGGCACATGCYTTCGT-3’(SEQ ID No.1)
COI-R1：5’-GGAAACACCTGCGAGGTGTA-3’(SEQ ID No.2)
COI-R2：5’-TARAGGATGGGATCGCCTCC-3’(SEQ ID No.3).
Wherein, base is mixed：R represents that A+G, Y represent C+T.
The beneficial effects of the invention are as follows：Simple and easy to do, specificity is high, can clearly differentiate and distinguish the similar little Huang of form
Fish and large yellow croaker juvenile fish.
Brief description of the drawings
Fig. 1 is gel electrophoresis figure of the invention.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, raw material and equipment for being used etc. is commercially available or commonly used in the art.
Method in following embodiments, unless otherwise instructed, is the conventional method of this area.
Embodiment：
First, DNA extractions are carried out using phenol/chloroform method：To with 95% alcohol or -20 DEG C of little yellow croakers of freezen protective and big
Yellow croaker sample, takes muscle of back tissue 50-100mg, using Traditional Method (phenol/chloroform extraction method) from extraction genomic DNA.
Method detailed and step are as follows：
(1) sample is put into 1.5ml centrifuge tubes to shred.Add 600 μ L STE (10mmol/L TriS-Cl, pH8.0；
0.1mol/L EDTA, pH 8.0；1%m/v SDS), it is uniform in concussion on oscillator.RNase (4 μ g/ μ L) 6ul is added, it is dense eventually
0.04 μ g/ μ L are spent, is incubated 1 hour in 37 DEG C of baking ovens.
(2) add Proteinase K (20 μ g/ μ L) 15 μ L, μ g/ μ l of final concentration 0.5, overturn and mix, be incubated in 50 DEG C of baking ovens
Night (＞ 8 hours).
(3) it is cool to room temperature after being taken out in baking oven：Plus 600 μ L (pH 8.0) balance phenols, it is gentle it is reverse mix to milkiness (>=
30min).Centrifugation (8000g, 10min), it is careful to draw supernatant to another clean centrifuge tube.
(4) isometric phenol is added：Chloroform：Isoamyl alcohol (25：24：1), overturn and mix 20min, be centrifuged (8000g, 10min),
It is careful to draw supernatant to another clean centrifuge tube.
(5) isometric chloroform is added：Isoamyl alcohol (24：1), overturn and mix 20min, be centrifuged (8000g, 10min), it is careful to draw
Supernatant is to another clean centrifuge tube.Isometric pre- cold isopropanol is added, is overturned and is mixed, -20 DEG C are deposited more than 2 hours.
(6) centrifugation (10000g, 10min) produces white precipitate, softly removes isopropanol, adds the pre- of 800 μ L75%
Cold alcohol, slow reverse washing DNA precipitations, then be centrifuged (10000g, 10min), abandon alcohol.
(7) (the note of repeat step 6：DNA precipitations should not be outwelled).
(8) DNA sample is placed in vacuum desiccator drying or how much oven drying (suitably adjusts depending on Residual ethanol in pipe
Drying time)；Add 100 μ l distilled waters to dissolve, put 4 DEG C of refrigerators it is stand-by or it is long-term be stored in -20 DEG C it is stand-by.
2nd, enter performing PCR using three special primers to the DNA for being extracted to expand.According to little yellow croaker in GenBank and greatly
The conserved sequence of the mitochondrial COI of yellow croaker carries out design of primers, and numbering is KU587530.1, DQ107813.1 in GenBank.
Primer sequence is as follows
COI-F：5’-GTTACGGCACATGCYTTCGT-3’
COI-R1：5 '-GGAAACACCTGCGAGGTGTA-3 ' (amplification 300bp bands)
COI-R2：5 '-TARAGGATGGGATCGCCTCC-3 ' (amplification 500bp bands).
Reaction system is as follows：
10 × buffer (buying from TAKARA companies) 2.5 μ l
dNTP(0.8mM) 2μl
COI-F(1μM) 1.0μl
COI-R1(1μM) 1.0μl
COI-R2(1μM) 1.0μl
Taq(0.04U/μl) 0.15μl
The μ l of DNA profiling 1
ddH2O supplies system to 25 μ l.
Reaction condition is：Reaction condition is 94 DEG C of predegeneration 3min, after by 40 circulations, each circulation includes 94 DEG C
45s, 50 DEG C of 45s, 72 DEG C of 45s, last 72 DEG C of extensions 10min.
Instrument is the type PCR instruments of Eppendorf Mastercycler 5333.
3rd, electrophoresis is carried out using agarose to PCR primer
Pcr amplification product is detected with 1.5% agarose gel electrophoresis.Electrophoresis during using Liuyi Instruments Plant, Beijing's bistable
Instrument (DYY-8C types), voltage is 140 volts, electrophoresis 20 minutes.Taken pictures detection using UVP GDS-8000 gel imaging systems.It is small
Yellow croaker sample probably amplifies a band in 500bp positions, and each appearance one probably 300bp and 500bp at of rheum officinale fish sample
Bar bright band, can be with quick discriminating little yellow croaker and large yellow croaker juvenile fish sample by banding pattern difference.
As shown in figure 1, after extracting amplification through DNA, in detected through gel electrophoresis result, what the left side was shown as 1 bright band is
The DNA cloning product of little yellow croaker, what centre was shown as 2 bright bands is the DNA cloning product of large yellow croaker, and the right is standard DNA mark
Note reference.Little yellow croaker and rheum officinale fish sample can be easily differentiated between using this method.
Embodiment described above is a kind of preferably scheme of the invention, not makees any formal to the present invention
Limitation, also has other variants and remodeling on the premise of without departing from the technical scheme described in claim.
SEQUENCE LISTING
<110>Zhejiang Ocean university
<120>A kind of little yellow croaker and the PCR authentication methods and primer of large yellow croaker juvenile fish
<130> 2017.03
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gttacggcac atgcyttcgt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggaaacacct gcgaggtgta 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
taraggatgg gatcgcctcc 20
Claims (5)
1. PCR authentication methods of a kind of little yellow croaker and large yellow croaker juvenile fish, it is characterised in that the PCR authentication methods step is：Carry
The genomic DNA of sample is taken, the DNA extracted using identification primer pair is entered performing PCR and expanded, pcr amplification product Ago-Gel
Electrophoresis detection, one amplified band of appearance is little yellow croaker, and two amplified bands of appearance are large yellow croaker；The identification primer sequence
Row are as follows：
COI-F：5’-GTTACGGCACATGCYTTCGT-3’
COI-R1：5’-GGAAACACCTGCGAGGTGTA-3’
COI-R2：5’-TARAGGATGGGATCGCCTCC-3’.
2. PCR authentication methods according to claim 1, it is characterised in that the PCR amplification system is：The PCR amplifications
System is：10 × buffer 2.5 μ l, dNTP 2 μ l, COI-F 1 μ l, COI-R1 1 μ l, COI-R2 1 μ l, the μ l of Taq enzyme 0.15,
DNA profiling 1 μ l, ddH2O supplies system to 25 μ l.
3. PCR authentication methods according to claim 2, it is characterised in that the final concentration of 0.8mM of dNTP, the end of COI-F
Concentration is 1 μM, final concentration of 1 μM of COI-R1, final concentration of 1 μM of COI-R2, the final concentration of 0.04U/ μ l of Taq enzyme.
4. PCR authentication methods according to claim 1 and 2, it is characterised in that the PCR amplification conditions are：94 DEG C of pre- changes
Property 3min, after by 40 circulations, each circulation includes 94 DEG C of 45s, 50 DEG C of 45s, 72 DEG C of 45s；Last 72 DEG C of extensions 10min.
5. a kind of primer for identifying little yellow croaker and large yellow croaker juvenile fish, it is characterised in that including three specific primers, primer
Sequence is as follows：
COI-F：5’-GTTACGGCACATGCYTTCGT-3’
COI-R1：5’-GGAAACACCTGCGAGGTGTA-3’
COI-R2：5’-TARAGGATGGGATCGCCTCC-3’.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828892A (en) * | 2017-11-02 | 2018-03-23 | 浙江海洋大学 | A kind of nonspecific immune reaction indicant of large yellow croaker |
| CN110684850A (en) * | 2019-09-04 | 2020-01-14 | 浙江海洋大学 | Primer sequence and method for rapidly identifying large and small yellow croakers |
| KR102254330B1 (en) * | 2019-11-18 | 2021-05-21 | 대한민국 | Primer set for determining identity of Miichthys miiuy and Sciaenops ocellatus, method of determining identity of Miichthys miiuy and Sciaenops ocellatus using the same and kit comprising the same |
| CN113215266A (en) * | 2021-04-14 | 2021-08-06 | 浙江省农业科学院 | Primer and method for rapidly identifying hybrid fish of large yellow croaker and small yellow croaker and parent thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107828892A (en) * | 2017-11-02 | 2018-03-23 | 浙江海洋大学 | A kind of nonspecific immune reaction indicant of large yellow croaker |
| CN110684850A (en) * | 2019-09-04 | 2020-01-14 | 浙江海洋大学 | Primer sequence and method for rapidly identifying large and small yellow croakers |
| KR102254330B1 (en) * | 2019-11-18 | 2021-05-21 | 대한민국 | Primer set for determining identity of Miichthys miiuy and Sciaenops ocellatus, method of determining identity of Miichthys miiuy and Sciaenops ocellatus using the same and kit comprising the same |
| CN113215266A (en) * | 2021-04-14 | 2021-08-06 | 浙江省农业科学院 | Primer and method for rapidly identifying hybrid fish of large yellow croaker and small yellow croaker and parent thereof |
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Application publication date: 20170623 |