CN108148916A - Sika deer identifies primer and methods and applications - Google Patents
Sika deer identifies primer and methods and applications Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- 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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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
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Abstract
The present invention relates to a kind of sika deer identification primer and methods and applications, belong to species identification technical field.The identification primer includes：Inner primer FIP：TGTCCTATACACCGATTTATGTGCTGCAAAACACGTGATATAACCT；Inner primer BIP：TGCCCCATGCTTATAAGCATGTGTACGATGAACAACATCATG；Outer primer F3：TCTATGTCCTACTAATTACAC；Outer primer B3：GACTGATTTGACTTAATGCAC；Ring primer LF：ACACGTGTGTCTTAATGTAC；Ring primer LB：TCCTATCATTTATAGTACATAGT.The primer is capable of the rapid amplifying sika deer nucleic acid of specificity, solves bottleneck problem of the pilose antler in " true and false " discriminating.
Description
Technical field
The present invention relates to species identification technical fields, and primer and methods and applications are identified more particularly to a kind of sika deer.
Background technology
Pilose antler refers to that the stag of sika deer is unossified and young horn with fine hair, is rare medicinal herbs.Contain phosphatide, sugar in pilose antler
Ingredients, the wherein aminoacid ingredient such as fat, glue fat, hormone, aliphatic acid, amino acid, protein and calcium, phosphorus, magnesium, sodium account for total ingredient
More than half.Li Shizhen (1518-1593 A.D.) exists《Compendium of Materia Medica》Upper title pilose antler " being good at tonifying kidney and strengthening yang, production of sperm benefit blood, mends marrow strong bone " is a kind of biography
The rare Chinese medicine of system.
But a large amount of velvet product true and false does not have to currently on the market, there is the problem on differentiating, there is an urgent need for it is a kind of quick,
Preferable, the reliable discrimination method for standardizing, standardizing.
2000, Notomi etc. invented ring mediated constant temperature nucleic acid amplification technology (loop-mediated isothermal
Amplification of DNA, LAMP), nucleic acid amplification can be realized under isothermal conditions.The reaction principle of LAMP technology is,
According to a pair of of outer primer that sequence preservative area is designed, six on a pair of of inner primer and a pair of of ring primer specificity identification target sequence
A isolated area, causes self-loopa strand replacement reaction under the action of Bst Large fragment polymerases, in 60~67 DEG C of range 60min,
It precipitates and generates with the magnesium pyrophosphate of by-product white while a large amount of synthesis target dnas.Since LAMP amplification procedures rely on
Identify six isolated areas of target sequence, so atopic is very strong, and amplification process is carried out under constant temperature,
Detection time greatly shortens, and quick sensitive.At present, LAMP technology is in bacterium and viral diagnosis, drug resistant gene detection, parasitism
Worm detection, sex identification etc. are widely used.
Invention content
Based on this, it is necessary in view of the above-mentioned problems, a kind of sika deer identification primer and method are provided, using the primer and side
Method can quickly examine sika deer nucleic acid, and the standardization identification for animal medicinal materials such as pilose antlers provides new thinking, solves pilose antler and exists
Bottleneck problem in " true and false " discriminating.
A kind of sika deer identifies primer, including following primer：
Inner primer FIP：TGTCCTATACACCGATTTATGTGCTGCAAAACACGTGATATAACCT；
Inner primer BIP：TGCCCCATGCTTATAAGCATGTGTACGATGAACAACATCATG；
Outer primer F3：TCTATGTCCTACTAATTACAC；
Outer primer B3：GACTGATTTGACTTAATGCAC；
Ring primer LF：ACACGTGTGTCTTAATGTAC；
Ring primer LB：TCCTATCATTTATAGTACATAGT.
Above-mentioned primer, be inventor according to the characteristics of sika deer genome, obtained after testing repeatedly, wherein, core
Region F1 (FIP back segments) and LF are placed to sika deer specific insert, ensure that the specificity of the set primer.The set primer
During for expanding non-sika deer nucleic acid, due to having lacked the segment, during amplification, cause FIP primers that cannot form circular dumbbell
Structure, therefore LAMP exponential amplifications can not be carried out, so as to which non-sika deer nucleic acid cannot be expanded.In addition, ring primer LF also is located at spy
The Insert Fragment of the opposite sex only only could carry out acceleration amplification when expanding sika deer nucleic acid, further ensure specificity.
In one of the embodiments, the working concentration of the inner primer FIP and BIP be 35-45pmol, the outer primer
The working concentration of F3 and B3 is 8-12pmol, and the working concentration of ring the primer LF and LB are 16-24pmol.Using above-mentioned concentration
Proportioning has preferable amplification efficiency.
The invention also discloses a kind of sika deer identification methods, include the following steps：
Ring mediated constant temperature nucleic acid amplification：It is expanded using the genome of above-mentioned primer pair sample to be tested；
Judgement：By measuring the turbidity of reaction solution, turbidity rises to positive reaction, i.e., the sample is sika deer sample, turbid
It is negative reaction to spend unchanged, i.e., the sample is non-sika deer sample；
And/or
Calcein indicator is added in into reaction solution, reaction solution is that green is positive reaction, i.e., the sample is sika deer
Sample, orange reaction solution is negative reaction, i.e., the sample is non-sika deer sample.
Method described above identifies sika deer kind have the advantages that specificity is high, quick and reliable.
In one of the embodiments, in the ring mediated constant temperature nucleic acid amplification step, amplification reaction system is 25 μ l, is wrapped
Include following component：Genomic DNA 2 μ l, the 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH of determinand4)2SO4, 0.1%Tween20,0.8M glycine betaines, 8mM MgSO4, 1.4mM dNTP each, 8U Bst DNA polymerase；
Primer addition is：40pmol inner primers FIP and inner primer BIP, 20pmol sequence table middle ring primer LF and ring draw
Object LB, 5pmol sequence table China and foreign countries primers F 3 and outer primer B3.
Using above-mentioned amplification system, there is preferable expanding effect.
In one of the embodiments, in the ring mediated constant temperature nucleic acid amplification step, the temperature of amplified reaction is 60-62
DEG C, the time of amplified reaction is 60-70min.Using above-mentioned temperature and time, there is preferable expanding effect.
The additive amount of calcein indicator is 1 μ l in the determination step in one of the embodiments, is included
0.5mM calceins and 10mM manganese chlorides.
The invention also discloses application of the above-mentioned sika deer identification primer in sika deer kind is identified.
The invention also discloses a kind of sika deer identification kit, including above-mentioned primer.
In one of the embodiments, the kit further include DNA extracts reagents, ring mediated constant temperature nucleic acid amplification reagent,
At least one of colored indicator and buffer solution.
The kit further includes positive control agent and negative control reagent in one of the embodiments,.
Compared with prior art, the invention has the advantages that：
A kind of sika deer identification primer of the present invention, can be accurate, quick and specific reflects to sika deer kind
Fixed, the standardization identification for animal medicinal materials such as pilose antlers provides new thinking, solves bottleneck of the pilose antler in " true and false " discriminating
Problem establishes the discrimination method of a kind of more quick, preferable, reliable standardization, standardization for pilose antler.
Description of the drawings
Fig. 1 is pattern detection turbidity-time changing curve figure in embodiment 1；
Wherein：Abscissa is the time, and ordinate is turbidity value；
Fig. 2 is pattern detection end turbidity figure in embodiment 1；
Fig. 3 is different dilution sample turbidity figures in embodiment 2；
Fig. 4 is that different temperatures expands sample turbidity figure in embodiment 3.
Specific embodiment
For the ease of understanding the present invention, the present invention is described more fully below with reference to relevant drawings.In attached drawing
Give presently preferred embodiments of the present invention.But the present invention can realize in many different forms, however it is not limited to this paper institutes
The embodiment of description.On the contrary, the purpose for providing these embodiments is to make the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The normally understood meaning of technical staff is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all combination of the Listed Items of pass.
Unless otherwise stated, agents useful for same is commercially available in following embodiment.
Embodiment 1
LAMP methods quickly detect the foundation of sika deer nuclei aoid methods
1 experimental program
1.1 material
Commercially available 12 parts of pilose antler samples (0740,0741,0742,0743,0744,0745,0746,0747,0748,
0749,0750,0751), wherein, 0748 is red deer sample, and 0749 is reinder sample, and add pig, duck, chicken, ox blood final proof
This is as sample to be tested.
1.2 instruments and reagent
LAMP method ribonucleic acid amplification kit (DNA Amplification Kit), Japanese Eiken Chemical
(EIKEN CHEMICAL CO.,LTD,Tochigi,Japan)；
LAMP reaction tubes (Reaction Tube), Japanese Eiken Chemical；
Visible dyes (Te), Beijing Haitai positive element bio tech ltd.
Real-time transmissometer La-320C, Japanese Eiken Chemical.
1.3 method
1.3.1Target the preparation of template target templates
Conventionally extract 12 parts of pilose antlers and pig respectively, duck, chicken, the nucleic acid in cow's serum is to get target template.
1.3.2LAMP the design of primer is reacted
Inventor has found, in sequence HQ832482.1 according to the characteristics of sika deer genome after comparing screening repeatedly
There are one section of insetion sequence (common 74bp, label is band underscore below) in (sika deer), as shown in the table, the segment is not
It is present in AB245426.1 and AB245427.2 (i.e. red deer and reinder).Therefore the sequence before and after the segment and the segment
Design LAMP primer.
The genome comparison of 1 sika deer of table, red deer and reinder
Primer is designed by distinguished sequence and context, destination region is following, and (underscore sequences in italics represents sika deer
Specific insert)：
Through analysis, sequence to be amplified is divided into eight independent regions, it is anti-to separately design LAMP according to this eight regions
6 required primers (inner primer FIP and BIP, outer primer F3 and B3, ring primer LF and LB) are answered, it is including but not limited to following to draw
Object combines：
Combination 1
F3:CTACTCAACATCCAATTTACA
FIP:GTGCTATTACACGTGTGTCTTAATG-GTCCTACTAATTACACAGC
BIP:CGGTGTATAGGACATATTATGTATAATAG-CATGCTTATAAGCATGGG
B3:GTACGATGAACAACATCATG
LF:CATATAAGGTTATATCACGTG
LB:GTATCAGGACATATTATGTATAATAG
Combination 2:
F3:TCTATGTCCTACTAATTACAC
FIP:TGTCCTATACACCGATTTATGTGCT-GCAAAACACGTGATATAACCTBIP:
TGCCCCATGCTTATAAGCATGT-GTACGATGAACAACATCATG
B3:GACTGATTTGACTTAATGCAC
LF:ACACGTGTGTCTTAATGTAC
LB:TCCTATCATTTATAGTACATAGT
Combination 3:
F3:CATGATGTTGTTCATCGTACA
FIP:AGCTCGTGATCTAGGGGACG-GTGCATTAAGTCAAATCAGTCC
BIP:GTGAAACCAGCAACCCGCTG-CTACCCCCACAATCCATG
B3:CCAGATGTCTGATAAAGTTCAT
LF:GGATACGCATGTTGACGA
LB:ATCCCTCTTCTCGCTCC
Combination 4:
F3:TGGACTAATGACTAATCAGCC
FIP:CCAAGCATCCCCCCAAAAATTAA-CATGCTCACACATAACTGTG
BIP:CTCAGCAATGGCCGTCTGAGG-CATTATGGGGATGCTCAAGA
B3:TGACTGCAATGCCCTACG
LF:AATACCAAATGTATGACACC
LB:TCCCGGAGCATGAATTGTA
Combination 5:
F3:GCCATCTCACCTAAAATCGC
FIP:
GTTATGTGTGAGCATGGGCTGA-CTCCTTGCAATATAAGACATCTCG
BIP:TTGGGGGGATGCTTGGACTC-CAATTCATGCTCCGGGAC
B3:GGATGCTCAAGATGCAGTT
LF:GTCCCGGAGCATGAATTG
LB:GAGTCCAAGCATCCCCCCAA
Combination 6:
F3:AGTACATAGTACATGATGTTGTTC
FIP:CTAGGGGACGGGATACGCAT-ATCGTACATAGTGCATTAAGTCABIP:
GTGAAACCAGCAACCCGCTG-CTACCCCCACAATCCATG
B3:CCAGATGTCTGATAAAGTTCAT
LF:CATGGATTGTGGGGGTAG
LB:CAGCGGGTTGCTGGTTTCAC
Combination 7:
F3:ACATCTGGTTCTTTTTTCAGG
FIP:TGGGCTGATTAGTCATTAGTCCATC-TCACCTAAAATCGCCCAC
BIP:TGCTTGGACTCAGCAATGGC-GTCCAGCTACAATTCATGC
B3:GGATGCTCAAGATGCAGTT
LF:GCATGAATTGTAGCTGGAC
LB:GCCATTGCTGAGTCCAAGCA
Combination 8:
F3:AATCAGCCCATGCTCACA
FIP:TGCTGAGTCCAAGCATCCCC-ATAACTGTGGTGTCATACATTTGG
BIP:GTCCCGGAGCATGAATTGTAG-TGACTGCAATGCCCTACG
B3:CATGAAATAATAACTATGTCCTGTG
LF:CGTAGGGCATTGCAGTCA
LB:CTACAATTCATGCTCCGGGAC
Combination 9:
F3:GGGGTAGCTATTTAATGAACTT
FIP:CGAGATGTCTTATATTGCAAGGAGT-CAGACATCTGGTTCTTTTTTCAG
BIP:CTAATCAGCCCATGCTCACACATAA-CCATTGCTGAGTCCAAGC
B3:GCTACAATTCATGCTCCGG
LF:GCTTGGACTCAGCAATGG
LB:TTATGTGTGAGCATGGGCTGATTAG
Combination 10:
F3:AAAATCGCCCACTCCTTG
FIP:CCAAATGTATGACACCACAGTTATG-GACATCTCGATGGACTAATGAC
BIP:TTGGGGGGATGCTTGGACTC-CAATTCATGCTCCGGGAC
B3:ATGCAGTTAAGTCCAGCT
LF:GTCCCGGAGCATGAATTG
LB:GAGTCCAAGCATCCCCCCAA
Combination 11:
F3:AACATGCGTATCCCGTCC
FIP:GAAGAGGGATCCCTGCCCAG-ATCACGAGCTTGATCACCA
BIP:CATGGATTGTGGGGGTAGCT-CCCTGAAAAAAGAACCAGATG
B3:AGTGGGCGATTTTAGGTG
LF:CATCTGGTTCTTTTTTCAGGG
LB:AGCTACCCCCACAATCCATG
Combination 12:
F3:GTTCATCGTACATAGTGCATTA
FIP:CATGGTGATCAAGCTCGTGAT-TCAAATCAGTCCTCGTCAA
BIP:GTGAAACCAGCAACCCGCTG-GCTACCCCCACAATCCAT
B3:CCAGATGTCTGATAAAGTTCAT
LF:ATGGATTGTGGGGGTAGC
LB:CAGCGGGTTGCTGGTTTCAC
Combination 13:
F3:GGGGTAGCTATTTAATGAACTT
FIP:AGTCATTAGTCCATCGAGATGTCTT-GTTCTTTTTTCAGGGCCATC
BIP:ATCAGCCCATGCTCACACATAAC-GCCATTGCTGAGTCCAAG
B3:GCTACAATTCATGCTCCGG
LF:CTTGGACTCAGCAATGGC
LB:GTTATGTGTGAGCATGGGCTGAT
Combination 14:
F3:CTTTATCAGACATCTGGTTCTT
FIP:AGTCATTAGTCCATCGAGATGTCTT-CAGGGCCATCTCACCTAA
BIP:ATCAGCCCATGCTCACACATAAC-GCCATTGCTGAGTCCAAG
B3:GCTACAATTCATGCTCCGG
LF:CTTGGACTCAGCAATGGC
LB:GTTATGTGTGAGCATGGGCTGAT
By a variety of trials and grope, contrived experiment compares after summing up experience, and 1 set of amplification efficiency of preferred design is good, amplification
Fireballing LAMP primer, for the amplification target gene of specificity.Finally obtain following primer pair：
Inner primer FIP：TGTCCTATACACCGATTTATGTGCT-GCAAAACACGTGATATAACCT(SEQ ID
No.1)
Inner primer BIP：TGCCCCATGCTTATAAGCATGT-GTACGATGAACAACATCATG(SEQ ID No.2)
Outer primer F3：TCTATGTCCTACTAATTACAC(SEQ ID No.3)
Outer primer B3：GACTGATTTGACTTAATGCAC(SEQ ID No.4)
Ring primer LF：ACACGTGTGTCTTAATGTAC(SEQ ID No.5)
Ring primer LB：TCCTATCATTTATAGTACATAGT(SEQ ID No.6).
Above-mentioned primer synthesis is synthesized and is purified by the farsighted biological Co., Ltd of Boxing science and technology in Beijing.
1.3.3LAMP reaction
LAMP reaction systems are 25 μ l, including：
Sample to be tested genomic DNA 2 μ l, 20mM TrisHCl (pH 8.8), 10mM KCl, 10mM (NH4) 2SO4,
0.1%Tween20,0.8M glycine betaine (betaine), 10mM MgSO4,1.4mMdNTP each, 8U Bst DNA
Polymerase (is purchased from NEB companies), and the primer amount of addition is：40pmol inner primers FIP and inner primer BIP, 20pmol ring draw
Object LF and ring primer LB, 5pmol outer primer F3 and outer primer B3.
React 12.5 μ l of buffer, 1 μ l Bst enzymes, 40pmol FIP and BIP, 10pmol F3 and B3,20pmol 528-
LF, 1 μ amplification colored indicator (Te).
Mixture is placed in 61 DEG C of isothermal reaction 65min, using distilled water as negative control.
1.3.4LAMP reaction result detects
A, real-time transmissometer detection：Formulas below occurs during the reaction for LAMP：
(DNA)n-1+dNTP—→(DNA)n+P2O7 4-
P2O7 4-+2Mg2+—→Mg2P2O7↓
Wherein Mg2P2O7That is magnesium pyrophosphate is white precipitate.According to this principle, Japanese Eiken Chemical's exploitation
Go out real-time transmissometer LA-320c, this experiment uses the transmissometer, and the turbidity for measuring reaction tube every 6 seconds is simultaneously depicted as curve
Judge the yin and yang attribute of reaction.
B, indicator detects：Added in reaction solution (the end reaction system be 26 μ l) of 1 μ l containing 0.5mM calceins and
The calcein color indicator of 10mM manganese chlorides, according to the color change judging result (principle of reaction solution：Calcein is gold
Belong to ion indicator, it can Mg in Indicator Reaction liquid2+Variation.), green represents that there are sika deer specific DNAs in sample to be tested
(positive), it is orange to represent that sika deer DNA (feminine gender) is not present in sample to be tested.
2. pattern detection result
As shown in Figs. 1-2, No. 1 to No. 10 sample standard deviation can be by the present embodiment as can be seen from the results for Turbidity measurement result
Method detected, and (sample 1 is respectively to sample 10：0740,0741,0742,0743,0744,0745,0746,0747,
0750,0751).No. 11 to No. 16 sample standard deviations cannot be detected that (sample 11 is respectively to sample 16 by LAMP method：0748,
0749, pig, duck, chicken, cow's serum).
The interpretation of indicator testing result is consistent with Turbidity measurement result.
These results suggest that the LAMP method of the present embodiment can identify sika deer nucleic acid, and with specificity
The advantages of good, efficient and convenient.
Embodiment 2
Sensitivity experiments.
It is (a concentration of to extract the obtained nucleic acid of sample 0743 in order to verify the detection sensitivity of the above method：76ng/μ
L) 10 doubling dilutions are done, altogether 7 dilution gradient (samples 0743,10-1, 10-2, 10-3, 10-4, 10-5, 10-6With 10-7), and with
Distilled water is as negative control.Then LAMP reactions are carried out according to the method for embodiment 1, knot is detected using nephelometry and Te
Fruit.
The results are shown in Figure 3, and LAMP can detect the 6th dilution of sample 0743, and nephelometry is tied with decoration method experiment
Fruit is consistent, and therefore, although the reaction time of set LAMP amplification systems is slightly slow, susceptibility is fabulous (because the can be detected
Six dilutions), it is 0.076pg/ μ l, which meets actually detected enough, and expands constant temperature time preferred 65min.
Embodiment 3
Optimum temperature is screened.
Optimum temperature screening is carried out to primer, temperature is from 59-66 DEG C, and gradient is 1 DEG C, then according to the method for embodiment 1
LAMP reactions are carried out, using nephelometry come testing result.
Testing result is as shown in figure 4, as can be seen from Fig., and LAMP reactions first have occurred in 60 DEG C, 61 DEG C, 62 DEG C whens, preferably
Temperature is 61 DEG C.
Embodiment 4
LAMP reactions are mixed object (identifying primer containing sika deer), Bst archaeal dna polymerases, distilled water, calcein
Indicator and positive control are packed jointly, obtain the sika deer identification kit of the present invention.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Embodiment described above only expresses the several embodiments of the present invention, and description is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that those of ordinary skill in the art are come
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Drug inspection research institute of Shenzhen
<120>Sika deer identifies primer and methods and applications
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213>Artificial sequence (Artificial)
<400> 1
tgtcctatac accgatttat gtgctgcaaa acacgtgata taacct 46
<210> 2
<211> 42
<212> DNA
<213>Artificial sequence (Artificial)
<400> 2
tgccccatgc ttataagcat gtgtacgatg aacaacatca tg 42
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence (Artificial)
<400> 3
tctatgtcct actaattaca c 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial)
<400> 4
gactgatttg acttaatgca c 21
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (Artificial)
<400> 5
acacgtgtgt cttaatgtac 20
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence (Artificial)
<400> 6
tcctatcatt tatagtacat agt 23
Claims (10)
1. a kind of sika deer identifies primer, which is characterized in that including following primer：
Inner primer FIP：TGTCCTATACACCGATTTATGTGCTGCAAAACACGTGATATAACCT；
Inner primer BIP：TGCCCCATGCTTATAAGCATGTGTACGATGAACAACATCATG；
Outer primer F3：TCTATGTCCTACTAATTACAC；
Outer primer B3：GACTGATTTGACTTAATGCAC；
Ring primer LF：ACACGTGTGTCTTAATGTAC；
Ring primer LB：TCCTATCATTTATAGTACATAGT.
2. sika deer according to claim 1 identifies primer, which is characterized in that the work of the inner primer FIP and BIP is dense
It spends for 35-45pmol, the working concentration of the outer primer F3 and B3 is 8-12pmol, the working concentration of ring the primer LF and LB
For 16-24pmol.
3. a kind of sika deer identification method, which is characterized in that include the following steps：
Ring mediated constant temperature nucleic acid amplification：It is carried out using the genome of claim 1-2 any one of them primer pair samples to be tested
Amplification；
Judgement：By measuring the turbidity of reaction solution, turbidity rises to positive reaction, i.e., the sample be sika deer sample, turbidity without
Change as negative reaction, i.e., the sample is non-sika deer sample；
And/or
Calcein indicator is added in into reaction solution, reaction solution is that green is positive reaction, i.e., the sample is sika deer sample,
Orange reaction solution is negative reaction, i.e., the sample is non-sika deer sample.
4. sika deer identification method according to claim 3, which is characterized in that the ring mediated constant temperature nucleic acid amplification step
In, amplification reaction system is 25 μ l, including following component：2 μ l, the 20mM TrisHCl (pH of genomic DNA of determinand
8.8), 10mM KCl, 10mM (NH4)2SO4, 0.1%Tween20,0.8M glycine betaines, 8mM MgSO4, 1.4mM dNTP each,
8U Bst DNA polymerase；
Primer addition is：Outside 40pmol inner primers FIP and inner primer BIP, 20pmol ring primer LF and ring primer LB, 5pmol
Primers F 3 and outer primer B3.
5. sika deer identification method according to claim 3, which is characterized in that the ring mediated constant temperature nucleic acid amplification step
In, the temperature of amplified reaction is 60-62 DEG C, and the time of amplified reaction is 60-70min.
6. sika deer identification method according to claim 3, which is characterized in that in the determination step, calcein refers to
The additive amount for showing agent is 1 μ l, includes 0.5mM calceins and 10mM manganese chlorides.
7. application of the sika deer identification primer described in claim 1 in sika deer kind is identified.
8. a kind of sika deer identification kit, it is characterised in that including primer described in claim 1.
9. sika deer identification kit according to claim 8, which is characterized in that further include DNA extracts reagents, ring mediation
At least one of constant temperature nucleic acid amplification reagent, colored indicator and buffer solution.
10. sika deer identification kit according to claim 9, which is characterized in that further include positive control agent and the moon
Property contrast agents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810132008.1A CN108148916A (en) | 2018-02-09 | 2018-02-09 | Sika deer identifies primer and methods and applications |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
| CN101974524A (en) * | 2010-11-23 | 2011-02-16 | 中国检验检疫科学研究院 | Primer, probe, kit and method used for authenticating truth of spotted deer antler |
| CN105838807A (en) * | 2016-05-18 | 2016-08-10 | 珠海出入境检验检疫局检验检疫技术中心 | Primer for LAMP detection method of sheep derived material, detection method and kit |
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2018
- 2018-02-09 CN CN201810132008.1A patent/CN108148916A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1932041A (en) * | 2005-07-06 | 2007-03-21 | 韩国韩医学研究院 | The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species |
| CN101974524A (en) * | 2010-11-23 | 2011-02-16 | 中国检验检疫科学研究院 | Primer, probe, kit and method used for authenticating truth of spotted deer antler |
| CN105838807A (en) * | 2016-05-18 | 2016-08-10 | 珠海出入境检验检疫局检验检疫技术中心 | Primer for LAMP detection method of sheep derived material, detection method and kit |
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| 刘海等: "中国大陆梅花鹿mtDNA控制区序列变异及种群遗传结构分析", 《动物学报》 * |
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Application publication date: 20180612 |