CN107858407A - A kind of PCR primer and its discrimination method for differentiating oligopeptide albumen - Google Patents
A kind of PCR primer and its discrimination method for differentiating oligopeptide albumen Download PDFInfo
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- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 51
- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 51
- 238000012850 discrimination method Methods 0.000 title abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 23
- 238000012408 PCR amplification Methods 0.000 claims abstract description 10
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 230000004087 circulation Effects 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 11
- 238000012163 sequencing technique Methods 0.000 claims description 11
- 239000000523 sample Substances 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 7
- 230000007850 degeneration Effects 0.000 claims description 7
- 238000000137 annealing Methods 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 6
- 230000036425 denaturation Effects 0.000 claims description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 5
- 230000000692 anti-sense effect Effects 0.000 claims description 5
- 239000012154 double-distilled water Substances 0.000 claims description 5
- 238000001962 electrophoresis Methods 0.000 claims description 5
- 239000008236 heating water Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 150000002989 phenols Chemical class 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 230000004044 response Effects 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 108010067770 Endopeptidase K Proteins 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 102000035195 Peptidases Human genes 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 235000019441 ethanol Nutrition 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 4
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 4
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 4
- 235000011151 potassium sulphates Nutrition 0.000 claims description 4
- 235000019833 protease Nutrition 0.000 claims description 4
- 238000002731 protein assay Methods 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical class OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 101100329008 Artemia franciscana COI gene Proteins 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 2
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- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 7
- 235000013305 food Nutrition 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 25
- 150000001413 amino acids Chemical class 0.000 description 7
- 241000965254 Apostichopus japonicus Species 0.000 description 4
- 241001441722 Takifugu rubripes Species 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
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- 238000003199 nucleic acid amplification method Methods 0.000 description 2
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- 241000894007 species Species 0.000 description 2
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- 241000251468 Actinopterygii Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000251511 Holothuroidea Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 235000001497 healthy food Nutrition 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
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- 230000035764 nutrition Effects 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
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- 230000008569 process Effects 0.000 description 1
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- 150000003384 small molecules Chemical class 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- 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
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Abstract
Field of food detection of the present invention, more particularly to a kind of PCR primer and its discrimination method for differentiating oligopeptide albumen.Primer sequence：5’‑ACTAATRCCTCCCTCCTT‑3’（Upstream）；5’‑ATAAACTTCTRGGTGTCC‑3’（Downstream）, wherein R=A or G.The present invention can utilize pair of primers to do not belong to together or the different subspecies of same species enter performing PCR amplification, accuracy rate is very high, overcome in the prior art oligomeric peptide product be difficult to identify and identify that detection method is excessively cumbersome, is difficult to the defects of large-scale popularization.
Description
Technical field
The invention belongs to field of food detection, more particularly to a kind of PCR primer for differentiating oligopeptide albumen and its discriminating side
Method.
Background technology
Small molecule active oligopeptide --- one by modern biotechnology scholar be referred to as " favorite of 21st century human health ",
The bioactive substance of the good reputation such as " happy magical angel ", " soul of life ", " most promising healthy food ".Biology is living
Property oligopeptide be usually obtained by protein degradation.Oligopeptide is intermediate product of the protein degradation into amino acid, is amino acid
Precursor substance, be formed by connecting by the polymerization of several or several amino acid moleculars, there is crude protein and single amino acid not to have
Standby unique physiologically active and medical care effect.It is used for food, health food, medical treatment, has nutrition, health care, treatment three
Weight function.
The peptide being made up of 2 ~ 10 amino acid is referred to as oligopeptide or oligopeptides, is referred to as by 10 peptides formed with upper amino acid more
Peptide.Existing a variety of oligomeric peptide products, but because the difference of enzymolysis process causes product quality uneven on the market at present.It is and low
In production, nucleic acid can be remained in oligopeptide finished product and can not removed poly- peptide.Because the molecular weight of peptide is low, and the peptide in peptide product
Chain is different in size, and amino acid composition is complicated, can not determine the source of peptide by the method that protein sequencing compares.In addition, by
In the albumen source detection difficult of peptide, there is illegal businessman to make oligopeptide by vegetable proteins such as soybean, to pretend to be ocean to give birth to
Thing such as sea cucumber, oyster oligopeptide are sold, and profit improves 50-60%, compromise the interests of consumer, and oligopeptide identification is difficult
Problem it is urgently to be resolved hurrily.
The content of the invention
It is an object of the invention to provide a kind of PCR primer and its discrimination method for differentiating oligopeptide albumen source, detection
Method cost is low, easy to operate.
The technical scheme taken to achieve the above object of the present invention is：A kind of PCR for differentiating oligopeptide albumen source draws
Thing, it is characterised in that the sequence of the primer is：
Sense primer：5’-ACTAATRCCTCCCTCCTT-3’
Anti-sense primer：5’-ATAAACTTCTRGGTGTCC-3’
Wherein R=A or G.
The primer is used to differentiate oligopeptide albumen source.
The primer is used to differentiate that reaction system during oligopeptide albumen source is：Containing Mg2+The μ of 10 × PCR buffer 5
L, dNTP 4 μ L, 10 μm of ol/L each 2 μ L of upstream and downstream primer, μ L, the 5U/ μ L of DNA profiling 2 TaqE 0.6 μ L, ddH2O
34.4μL。
The primer is used to differentiate that response parameter during oligopeptide albumen source is：94 DEG C of pre-degeneration 5min, subsequently into
Circulation, 94 DEG C of denaturation 45s, 48 DEG C of annealing 45s, 72 DEG C of extension 60s, totally 35 circulate, final 72 DEG C of extensions 10min.
A kind of method for differentiating oligopeptide albumen using the PCR primer, it is characterised in that comprise the following steps：
(1) sample pretreatment
1. taking the 2.5ml centrifuge tubes of sterilizing, oligomeric peptide product 0.5-2.5g is put into；
2. fully mixed after adding 500-700ml DNA extract solutions and 5-20 μ L Proteinase Ks in centrifuge tube；
3. centrifuge tube is put into heating water bath in 50-60 DEG C of water-bath, 15 min upsets are often crossed once, heating 2-6h takes out, and adds
Isometric saturated phenol with DNA extract solutions, lower extracting 10min is jiggled, then with 12000rpm, centrifuge 10min；
4. supernatant add with the isometric phenol/chloroform mixed liquor of DNA extract solutions softly mix 10min, then with
12000rpm centrifuges 10min；
5. supernatant adds softly mixes 10min with the isometric chloroform of DNA extract solutions, then centrifuged with 12000 rpm
10min；
6. supernatant adds the absolute ethyl alcohol of 2 times of DNA extracting liquid volumes, turn-take and shake up, 10min is centrifuged with 12000 rpm；
7. abandoning supernatant, washed with 70% ethanol after precipitating 2-3 times, normal temperature dries；
8. adding the dissolving of 50-150 μ L TE solution, -20 DEG C save backup；
（2）PCR amplifications, sequencing
Primer sequence：Sense primer：5’-ACTAATRCCTCCCTCCTT-3’
Anti-sense primer：5’-ATAAACTTCTRGGTGTCC-3’
Wherein R=A or G；
Reaction system is prepared using above-mentioned primer, in the enterprising performing PCR amplification of PCR instrument, response parameter is：94 DEG C of pre-degenerations
5min, subsequently into circulation, 94 DEG C of denaturation 45s, 48 DEG C of annealing 45s, 72 DEG C of extension 60s, totally 35 circulations, final 72 DEG C extend
10min, the fragment amplified is subjected to electrophoresis checking, is then sequenced；
（3）Genetic Distance Analysis
Sequencing result is compared with NCBI CO I genes storehouse, similarity is drawn, when similarity result is more than 99.5%
The source of species oligopeptide is can be identified as, and what subspecies raw material under which kind of can be differentiated；
（4）Quality Identification
1. weighing oligopeptide sample 2g, 10mL 9-15% trichloroacetic acids are added, is well mixed and stands 10min；Sample solution is existed
Whole supernatants are taken after centrifuging 10min under 4000r/min；
2. add copper sulphate：Potassium sulfate is 1：30 mixture 2.6g and the 10mL concentrated sulfuric acid, is put on digesting and digests, 200 DEG C
After predigestion 1h 430 DEG C digestion 3h, to digest tube in solution be changed into blue-tinted transparent；
3. room temperature is put in cooling, routinely protein assay method distills in kjeldahl apparatus and carries out result calculating, and protein changes
Calculate coefficient and take 5.79.
The step（1）- 2. middle DNA extract solutions be：The 10mmol/L Tris-Cl of pH=8.0, the 100mmol/ of pH=8.0
L EDTA, the SDS of quality volume fraction 10%；Proteinase K is：200mg Proteinase K adds 9.5ml distilled waters, is settled to
20ml。
The step（1）- 8. middle TE solution be：The 10mmol/L Tris-Cl of pH=8.0, the 100mmol/L of pH=8.0
EDTA。
The step（2）Middle PCR reaction systems are：Containing Mg2+10 × PCR buffer 5 μ L, dNTP 4 μ L, 10 μm of ol/
L each 2 μ L of upstream and downstream primer, μ L, the 5U/ μ L of DNA profiling 2 TaqE 0.6 μ L, ddH2O 34.4μL。
The beneficial effects of the present invention are：
1st, oligopeptide identification of the present invention and detection method are simple and quick using PCR method, can be fast and accurately
Identify oligopeptide and distinguish oligopeptide and the identification of other peptide products；
2nd, CO I genes of the present invention are that chondriogen has conservative, and evolutionary rate is moderate, rich in development signal etc.
Feature, can utilize pair of primers to do not belong to together or the different subspecies of same species enter performing PCR amplification, accuracy rate is very high, gram
Oligomeric peptide product is difficult to identify and identifies that detection method is excessively cumbersome, is difficult to the defects of large-scale popularization clothes in the prior art.
Brief description of the drawings
Fig. 1 is electrophoretogram of the embodiment of the present invention；
Wherein：M is DL5000marker, and for A to differentiate imitative stichopus japonicus oligopeptide albumen amplification, B is to differentiate that Fugu rubripes is low
Poly- peptide albumen amplification.
Embodiment
The present invention is further described with embodiment below in conjunction with the accompanying drawings, but the present invention is not limited to specific embodiment.
Embodiment 1
A kind of PCR primer for differentiating oligopeptide albumen source, its sequence are：
Sense primer：5’-ACTAATRCCTCCCTCCTT-3’
Anti-sense primer：5’-ATAAACTTCTRGGTGTCC-3’
Wherein R=A or G.
The primer is used to differentiate oligopeptide albumen source, and reaction system is：Containing Mg2+The μ L of 10 × PCR buffer 5,
DNTP 4 μ L, 10 μm of ol/L each 2 μ L of upstream and downstream primer, μ L, the 5U/ μ L of DNA profiling 2 TaqE 0.6 μ L, ddH2O 34.4μ
L；Response parameter is：94 DEG C of pre-degeneration 5min, subsequently into circulation, 94 DEG C of denaturation 45s, 48 DEG C of annealing 45s, 72 DEG C extend 60s,
Totally 35 circulations, final 72 DEG C of extensions 10min.
Embodiment 2
A kind of method for differentiating oligopeptide albumen using the specificity of PCR primer described in embodiment 1, sample nucleic in the present embodiment
Sequence specifically includes following steps as shown in sequence 3 in sequence table：
(1) sample pretreatment：
1. taking the 2.5mL centrifuge tubes of sterilizing, oligomeric peptide product 1g is put into.
2. fully mixed after adding 600mL DNA extract solutions and 10 μ L Proteinase Ks in centrifuge tube.
3. centrifuge tube is put into heating water bath in 55 DEG C of water-baths, often crosses 15 min upsets once, 4h, take out and add 600mL
Saturated phenol, extracting（Jiggle）10min.Then with 12000rpm, 10min is centrifuged.
4. supernatant adds 600mL phenol/chloroform mixed liquors and softly mixes 10min, then centrifuged with 12000rpm
10min。
5. supernatant adds 600mL chloroforms and softly mixes 10min, then 10min is centrifuged with 12000 rpm.
6. supernatant adds 1200mL absolute ethyl alcohol（Turn-take and shake up）, 10min is centrifuged with 12000 rpm.
7. abandon supernatant with 70% ethanol wash precipitation 2 times after, normal temperature dries.
8. add the dissolving of 100 μ L TE solution.
(2) PCR amplifications, sequencing
Enter performing PCR amplification using primer described in embodiment 1.PCR reaction systems are：10×PCR buffer（Containing Mg2+）5 μ L,
DNTP4 μ L, upstream and downstream primer（10μmol/L）Each 2 μ L, DNA profiling 2 μ L, TaqE（5U/μL）0.6 μ L, ddH2The μ L of O 34.4,
Reaction solution is prepared in PCR instrument through 94 DEG C of pre-degeneration 5min, subsequently into circulation, 94 DEG C of denaturation 45s, 48 DEG C of 45s that anneal, 72
DEG C extension 60s, totally 35 circulation, it is final 72 DEG C extension 10min, by the fragment amplified carry out electrophoresis checking（The result is shown in
A bands in Fig. 1）, then it is sequenced.
(3) Genetic Distance Analysis
Sequencing result is subjected to sequence alignment analysis in NCBI.
（4）Quality Identification
1. weighing oligopeptide sample 2g, the trichloroacetic acids of 10mL 15% are added, is well mixed and stands 10min.Sample solution is existed
Whole supernatants are taken after centrifuging 10min under 4000r/min.
2. add copper sulphate：Potassium sulfate（1：30）2.6g the and 10mL concentrated sulfuric acids, it is put on digesting and digests, 200 DEG C disappears in advance
Change 1h, 430 DEG C of digestion 3h to solution in digest tube are changed into blue-tinted transparent.
3. room temperature is put in cooling, routinely protein assay method distills in kjeldahl apparatus and carries out result calculating, albumen
Matter conversion coefficient takes 5.79.
It is imitative stichopus japonicus to compare analysis oligopeptide rule of origin according to sequencing gene（Such as sequence table of Apostichopus japonicus standard nucleotide sequence
Shown in middle sequence 5）, oligomeric peptide content reaches 90%.
Embodiment 3
A kind of method for differentiating oligopeptide albumen using the specificity of PCR primer described in embodiment 1, sample nucleic in the present embodiment
Sequence specifically includes following steps as shown in sequence 4 in sequence table：
(1) sample pretreatment：
1. taking the 2.5ml centrifuge tubes of sterilizing, ocean fish oligopeptide product 0.5g is put into.
2. fully mixed after adding 500mlDNA extract solutions and 5 μ L Proteinase Ks in centrifuge tube.
3. centrifuge tube is put into heating water bath in 50 DEG C of water-baths, often crosses 15 min upsets once, 2h, take out and add 500ml
Saturated phenol, extracting（Jiggle）10min.Then with 12000rpm, 10min is centrifuged.
4. supernatant adds 500ml phenol/chloroform mixed liquors and softly mixes 10min, then centrifuged with 12000rpm
10min。
5. supernatant adds 500ml chloroforms and softly mixes 10min, then 10min is centrifuged with 12000 rpm.
6. supernatant adds 1000ml absolute ethyl alcohol（Turn-take and shake up）, 10min is centrifuged with 12000 rpm.
7. abandon supernatant with 70% ethanol wash precipitation 2 times after, normal temperature dries.
8. add the dissolving of 50 μ L TE solution.
(2) PCR amplifications, sequencing
Enter performing PCR amplification using primer described in embodiment 1.Preparing PCR reaction systems is：10×PCR buffer（Mg2+
Plus）5 μ L, dNTP4 μ L, upstream and downstream primer (10 μm of ol/L) each 2 μ L, DNA profiling 2 μ L, TaqE（5U/μL）0.6 μ L,
DdH2O34.4 μ L, reaction solution is prepared in PCR instrument through 94 DEG C of pre-degeneration 5min, subsequently into circulation, 94 DEG C of denaturation 45s, 48
DEG C annealing 45s, 72 DEG C extension 60s, totally 35 circulation, it is final 72 DEG C extension 10min, by the fragment amplified carry out electrophoresis checking
（The B bands that the result is shown in Fig. 1）, then it is sequenced.
(3) Genetic Distance Analysis
By sequencing result after artificial check and correction and splicing, sequence alignment analysis is carried out in NCBI blast.
（4）Quality Identification
1. weighing oligopeptide sample 2g, the trichloroacetic acids of 10mL 15% are added, is well mixed and stands 10min.Sample solution is existed
Whole supernatants are taken after centrifuging 10min under 4000r/min.
2. add copper sulphate：Potassium sulfate（1：30）2.6g the and 10mL concentrated sulfuric acids, it is put on digesting and digests, 200 DEG C disappears in advance
Change 1h, 430 DEG C of digestion 3h to solution in digest tube are changed into blue-tinted transparent.
3. room temperature is put in cooling, routinely protein assay method distills in kjeldahl apparatus and carries out result calculating, albumen
Matter conversion coefficient takes 5.79.
It is Fugu rubripes to compare analysis oligopeptide rule of origin according to sequencing gene（Fugu rubripes standard nucleic acid sequence
As shown in sequence 6 in sequence table）, oligomeric peptide content reaches 85%.
Embodiment 4
Described in the present embodiment utilize embodiment 1 in PCR primer specificity differentiate oligopeptide albumen method each step with
Embodiment 2 or 3 is identical, and different parameters is：
Step（1）In：
1）Oligomeric peptide product 2.5g is put into centrifuge tube.
2）It is 700ml that DNA extract solutions, saturated phenol, phenol/chloroform mixed liquor, the volume of chloroform are added in centrifuge tube, is added
The volume of absolute ethyl alcohol is 1000ml.
3）Centrifuge tube is put into heating water bath 6h in 60 DEG C of water-baths.
4）Add the dissolving of 150 μ L TE solution.
5）It is imitative stichopus japonicus to compare analysis oligopeptide rule of origin according to sequencing gene, and oligomeric peptide content reaches 90%.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope of present disclosure, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Sequence table
SEQUENCE LISTING
<110>The dark blue peptide science and technology research and development Co., Ltd in Dalian
<120>A kind of PCR primer and its discrimination method for differentiating oligopeptide albumen
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> Artificial
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<221> misc_feature
<222> (7)..(7)
<223>R is a or g
<400> 1
actaatrcct ccctcctt 18
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<211> 18
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (11)..(11)
<223>R is a or g
<400> 2
ataaacttct rggtgtcc 18
<210> 3
<211> 485
<212> DNA
<213> Artificial
<400> 3
tgaactgttt acccacccct agcaggaaat cttgcccacg caggtacttc tgtagacctc 60
accatcttct ctcttcatct tgcaggggtc tcctctattc taggttcgat caacttcatc 120
acaactatca ttaacatgaa gcccccagca atcacacaat accaaatcct cttatcatta 180
tagatccgta tttataactg ctatcctttt acttctgagc cttccagtac tagctggagc 240
cataacgatg ttactaacgg accgtaaaat taaaacaact ttttttgacc cagcaggtgg 300
aggagaccca atattgtttc aacacttgtt ctgattcttc ggacacccag aagcttatat 360
tttaatacta cctggatttg gtatgatctc tcacgctata gcacattata gaggtaagca 420
agaacccttc ggttatttag gtatggtgta tgccatgggg gctataggga tactaggatt 480
tcttg 485
<210> 4
<211> 659
<212> DNA
<213> Artificial
<400> 4
cggtatgcat tatgacgata gctcagacat gtccttcccc cgaatgaaca acatagagca 60
ttctgactgc ttcccccatc cttcctcctt ctgctcgcat cctctggagt agaagccgga 120
gcgggaacgg gctgaactgt ttacccaccc ctagcaggaa atcttgccca cgcaggagct 180
tctgtagacc tcaccatctt ctctcttcat cttgcagggg tctcctctat tctaggttcg 240
atcaacttca tcacaact at cattaacatg aagcccccag caatctcaca ataccaaaca 300
cctctattcg tgtgagccgt attaattact gctgtacttc tcctgctctc ccttccagtc 360
ctagcagcag ggattacaat acttctcact gaccgaaacc taaatacaac cttctttgac 420
ccagcaggag gaggagaacc catcttgtac caacacttat tctgaatctt tggacaccct 480
gaagtctaca ttctaattct ccctggcttc ggaataaatc cccacatcga aacctactac 540
tcgagcaaaa aggaacattc ggctacatga gcatggtctg agccataatg gccatcgggc 600
ttcttagttt attggtgagc ccaccctatt tacaggtgga agaataccac ccaaacaaa 659
<210> 5
<211> 652
<212> DNA
<213> Artificial
<400> 5
taaatgatta attcctctaa tgataggtgc tccagacatg gctttcccac gaatgaaaaa 60
aatgagattt tgactaatac ctccctcctt cattcttctt cttgcctctg caggagttga 120
aagaggggcc ggaacagggt gaacaattta ccctccactc tcaagcaata ttgcccacgc 180
aggaggatct gttgacctag ctattttttc actacacttg gctggtgcct cctcaattct 240
agcttccata aaattcataa ccacaattat taaaatgcgg actccaggga taacttttga 300
tcgacttccc ttattcgtgt gatccgtatt tataactgct attcttttac ttctgagcct 360
tccagtacta gctggagcca taacgatgtt actaacggac cgtaaaatta aaacaacttt 420
ttttgaccca gcaggtggag gagacccaat attgtttcaa cacttgttct gattcttcgg 480
acacccagaa gtttatattt taatactacc tggatttggt atgatctctc acgttatagc 540
acattataga ggtaagcaag aacccttcgg ttatttaggt atggtgtatg ccatggtggc 600
tatagggata ctaggatttc ttgtttgggc ccaccatatg tttactgttg gt 652
<210> 6
<211> 1546
<212> DNA
<213> Artificial
<400> 6
gtggcaatca cacgctgatt tttctcaacc aatcacaaag atatcggcac cctataccta 60
gtttttggtg cctgagccgg aatagtaggc acagcactaa gtcttcttat tcgggccgaa 120
ctcagtcaac ccggcgcact cttgggcgat gaccagattt acaatgtaat cgttacagcc 180
catgcattcg taatgatttt ctttatagta ataccaatca tgattggagg ctttggaaac 240
tgattagttc ccctaataat tggagcccca gacatggcct tcccccgaat aaacaacata 300
agcttctgac tgcttccccc atccttcctc cttctactcg catcctctgg agtagaagcc 360
ggagcgggta cgggctgaac cgtttaccca cccctagcag gaaatcttgc ccacgcagga 420
gcttctgtag accttaccat cttctctctt catcttgcag gggtctcctc tattctaggg 480
gcaatcaact tcatcacaac tatcattaac atgaaacccc cagcaatctc acaataccaa 540
acaccccttt tcgtgtgagc cgtcttaatt actgctgtac ttctcctgct ctcccttcca 600
gtacttgcag cagggattac aatacttctc actgaccgaa acctaaatac aaccttcttt 660
gacccagcag gaggaggaga ccccatcctg taccaacact tattctgatt ctttgggcac 720
cctgaagtct acattctaat tctccccggc ttcggaataa tctcgcacat cgtagcctac 780
tactcgggca aaaaggaacc attcggttac atgggcatgg tatgagccat gatggccatc 840
ggtcttcttg gctttattgt atgagcccac cacatgttta cagttggcat ggacgtagac 900
acccgagcct actttacctc cgccacaata attattgcca tcccaacagg ggtaaaagta 960
ttcagttgac ttgcaacctt gcatggagga tcaattaaat gagaaacccc tatactatgg 1020
gccctcggct tcatcttcct atttacagtg ggtggcctaa ccggaattgt cctggccaac 1080
tcatccctag acattgtgtt acacgacacc tactacgtag ttgcccattt ccactatgtc 1140
ctctccatgg gtgctgtatt tgcaatcatg ggtgcattcg tacactgatt cccactattc 1200
tcaggataca cactccacag tacttgaact aaaattcact tcggagtaat gtttattggt 1260
gtcaacctaa ccttcttccc tcaacacttc cttggcctag ctgggatacc tcgacgatat 1320
tccgactacc ccgacgccta cgccctatga aactctgtct cctcaattgg atcaatagtc 1380
tctctagtgg cagttattat gttcctcttt atcctctgag aagccttcac cgctaagcga 1440
gaagtccaat cagttgaact aacaatgaca aatgtagaat gactacacgg gtgccctcct 1500
ccctaccaca catttgaaga acccgccttc gttcaaactc aaacct 1546
SEQUENCE LISTING
<110>The dark blue peptide science and technology research and development Co., Ltd in Dalian
<120>A kind of PCR primer and its discrimination method for differentiating oligopeptide albumen
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (7)..(7)
<223>R is a or g
<400> 1
actaatrcct ccctcctt 18
<210> 2
<211> 18
<212> DNA
<213> Artificial
<220>
<221> misc_feature
<222> (11)..(11)
<223>R is a or g
<400> 2
ataaacttct rggtgtcc 18
<210> 3
<211> 485
<212> DNA
<213> Artificial
<400> 3
tgaactgttt acccacccct agcaggaaat cttgcccacg caggtacttc tgtagacctc 60
accatcttct ctcttcatct tgcaggggtc tcctctattc taggttcgat caacttcatc 120
acaactatca ttaacatgaa gcccccagca atcacacaat accaaatcct cttatcatta 180
tagatccgta tttataactg ctatcctttt acttctgagc cttccagtac tagctggagc 240
cataacgatg ttactaacgg accgtaaaat taaaacaact ttttttgacc cagcaggtgg 300
aggagaccca atattgtttc aacacttgtt ctgattcttc ggacacccag aagcttatat 360
tttaatacta cctggatttg gtatgatctc tcacgctata gcacattata gaggtaagca 420
agaacccttc ggttatttag gtatggtgta tgccatgggg gctataggga tactaggatt 480
tcttg 485
<210> 4
<211> 659
<212> DNA
<213> Artificial
<400> 4
cggtatgcat tatgacgata gctcagacat gtccttcccc cgaatgaaca acatagagca 60
ttctgactgc ttcccccatc cttcctcctt ctgctcgcat cctctggagt agaagccgga 120
gcgggaacgg gctgaactgt ttacccaccc ctagcaggaa atcttgccca cgcaggagct 180
tctgtagacc tcaccatctt ctctcttcat cttgcagggg tctcctctat tctaggttcg 240
atcaacttca tcacaactat cattaacatg aagcccccag caatctcaca ataccaaaca 300
cctctattcg tgtgagccgt attaattact gctgtacttc tcctgctctc ccttccagtc 360
ctagcagcag ggattacaat acttctcact gaccgaaacc taaatacaac cttctttgac 420
ccagcaggag gaggagaacc catcttgtac caacacttat tctgaatctt tggacaccct 480
gaagtctaca ttctaattct ccctggcttc ggaataaatc cccacatcga aacctactac 540
tcgagcaaaa aggaacattc ggctacatga gcatggtctg agccataatg gccatcgggc 600
ttcttagttt attggtgagc ccaccctatt tacaggtgga agaataccac ccaaacaaa 659
<210> 5
<211> 652
<212> DNA
<213> Artificial
<400> 5
taaatgatta attcctctaa tgataggtgc tccagacatg gctttcccac gaatgaaaaa 60
aatgagattt tgactaatac ctccctcctt cattcttctt cttgcctctg caggagttga 120
aagaggggcc ggaacagggt gaacaattta ccctccactc tcaagcaata ttgcccacgc 180
aggaggatct gttgacctag ctattttttc actacacttg gctggtgcct cctcaattct 240
agcttccata aaattcataa ccacaattat taaaatgcgg actccaggga taacttttga 300
tcgacttccc ttattcgtgt gatccgtatt tataactgct attcttttac ttctgagcct 360
tccagtacta gctggagcca taacgatgtt actaacggac cgtaaaatta aaacaacttt 420
ttttgaccca gcaggtggag gagacccaat attgtttcaa cacttgttct gattcttcgg 480
acacccagaa gtttatattt taatactacc tggatttggt atgatctctc acgttatagc 540
acattataga ggtaagcaag aacccttcgg ttatttaggt atggtgtatg ccatggtggc 600
tatagggata ctaggatttc ttgtttgggc ccaccatatg tttactgttg gt 652
<210> 6
<211> 1546
<212> DNA
<213> Artificial
<400> 6
gtggcaatca cacgctgatt tttctcaacc aatcacaaag atatcggcac cctataccta 60
gtttttggtg cctgagccgg aatagtaggc acagcactaa gtcttcttat tcgggccgaa 120
ctcagtcaac ccggcgcact cttgggcgat gaccagattt acaatgtaat cgttacagcc 180
catgcattcg taatgatttt ctttatagta ataccaatca tgattggagg ctttggaaac 240
tgattagttc ccctaataat tggagcccca gacatggcct tcccccgaat aaacaacata 300
agcttctgac tgcttccccc atccttcctc cttctactcg catcctctgg agtagaagcc 360
ggagcgggta cgggctgaac cgtttaccca cccctagcag gaaatcttgc ccacgcagga 420
gcttctgtag accttaccat cttctctctt catcttgcag gggtctcctc tattctaggg 480
gcaatcaact tcatcacaac tatcattaac atgaaacccc cagcaatctc acaataccaa 540
acaccccttt tcgtgtgagc cgtcttaatt actgctgtac ttctcctgct ctcccttcca 600
gtacttgcag cagggattac aatacttctc actgaccgaa acctaaatac aaccttcttt 660
gacccagcag gaggaggaga ccccatcctg taccaacact tattctgatt ctttgggcac 720
cctgaagtct acattctaat tctccccggc ttcggaataa tctcgcacat cgtagcctac 780
tactcgggca aaaaggaacc attcggttac atgggcatgg tatgagccat gatggccatc 840
ggtcttcttg gctttattgt atgagcccac cacatgttta cagttggcat ggacgtagac 900
acccgagcct actttacctc cgccacaata attattgcca tcccaacagg ggtaaaagta 960
ttcagttgac ttgcaacctt gcatggagga tcaattaaat gagaaacccc tatactatgg 1020
gccctcggct tcatcttcct atttacagtg ggtggcctaa ccggaattgt cctggccaac 1080
tcatccctag acattgtgtt acacgacacc tactacgtag ttgcccattt ccactatgtc 1140
ctctccatgg gtgctgtatt tgcaatcatg ggtgcattcg tacactgatt cccactattc 1200
tcaggataca cactccacag tacttgaact aaaattcact tcggagtaat gtttattggt 1260
gtcaacctaa ccttcttccc tcaacacttc cttggcctag ctgggatacc tcgacgatat 1320
tccgactacc ccgacgccta cgccctatga aactctgtct cctcaattgg atcaatagtc 1380
tctctagtgg cagttattat gttcctcttt atcctctgag aagccttcac cgctaagcga 1440
gaagtccaat cagttgaact aacaatgaca aatgtagaat gactacacgg gtgccctcct 1500
ccctaccaca catttgaaga acccgccttc gttcaaactc aaacct 1546
Claims (8)
1. a kind of PCR primer for differentiating oligopeptide albumen source, it is characterised in that the sequence of the primer is：
Sense primer：5’-ACTAATRCCTCCCTCCTT-3’
Anti-sense primer：5’-ATAAACTTCTRGGTGTCC-3’
Wherein R=A or G.
A kind of 2. PCR primer for differentiating oligopeptide albumen source according to claim 1, it is characterised in that the primer
Differentiate oligopeptide albumen source for specificity.
3. a kind of PCR primer for differentiating oligopeptide albumen source according to claim 1 or 2, it is characterised in that described to draw
Thing is used for specificity and differentiates that reaction system during oligopeptide albumen source is：Containing Mg2+The μ L, dNTP 4 of 10 × PCR buffer 5
μ L, 10 μm of ol/L each 2 μ L of upstream and downstream primer, μ L, the 5U/ μ L of DNA profiling 2 TaqE 0.6 μ L, ddH2O 34.4μL。
4. a kind of PCR primer for differentiating oligopeptide albumen source according to claim 1 or 2, it is characterised in that described to draw
Thing is used for specificity and differentiates that response parameter during oligopeptide albumen source is：94 DEG C of pre-degeneration 5min, subsequently into circulation, 94 DEG C
45s is denatured, 48 DEG C of annealing 45s, 72 DEG C of extension 60s, totally 35 circulations, final 72 DEG C extend 10min.
A kind of 5. method for differentiating oligopeptide albumen using PCR primer described in claim 1, it is characterised in that including following step
Suddenly：
(1) sample pretreatment
1. taking the 2.5ml centrifuge tubes of sterilizing, oligomeric peptide product 0.5-2.5g is put into；
2. fully mixed after adding 500-700ml DNA extract solutions and 5-20 μ L Proteinase Ks in centrifuge tube；
3. centrifuge tube is put into heating water bath in 50-60 DEG C of water-bath, 15 min upsets are often crossed once, heating 2-6h takes out, and adds
Isometric saturated phenol with DNA extract solutions, lower extracting 10min is jiggled, then with 12000rpm, centrifuge 10min；
4. supernatant add with the isometric phenol/chloroform mixed liquor of DNA extract solutions softly mix 10min, then with
12000rpm centrifuges 10min；
5. supernatant adds softly mixes 10min with the isometric chloroform of DNA extract solutions, then centrifuged with 12000 rpm
10min；
6. supernatant adds the absolute ethyl alcohol of 2 times of DNA extracting liquid volumes, turn-take and shake up, 10min is centrifuged with 12000 rpm；
7. abandoning supernatant, washed with 70% ethanol after precipitating 2-3 times, normal temperature dries；
8. adding the dissolving of 50-150 μ L TE solution, -20 DEG C save backup；
（2）PCR amplifications, sequencing
Primer sequence：Sense primer：5’-ACTAATRCCTCCCTCCTT-3’
Anti-sense primer：5’-ATAAACTTCTRGGTGTCC-3’
Wherein R=A or G；
Reaction system is prepared using above-mentioned primer, in the enterprising performing PCR amplification of PCR instrument, response parameter is：94 DEG C of pre-degenerations
5min, subsequently into circulation, 94 DEG C of denaturation 45s, 48 DEG C of annealing 45s, 72 DEG C of extension 60s, totally 35 circulations, final 72 DEG C extend
10min, the fragment amplified is subjected to electrophoresis checking, is then sequenced；
（3）Genetic Distance Analysis
Sequencing result is compared with NCBI CO I genes storehouse, similarity is drawn, when similarity result is more than 99.5%
The source of species oligopeptide is can be identified as, and what subspecies raw material under which kind of can be differentiated；
（4）Quality Identification
1. weighing oligopeptide sample 2g, 10mL 9-15% trichloroacetic acids are added, is well mixed and stands 10min；Sample solution is existed
Whole supernatants are taken after centrifuging 10min under 4000r/min；
2. add copper sulphate：Potassium sulfate is 1：30 mixture 2.6g and the 10mL concentrated sulfuric acid, is put on digesting and digests, 200 DEG C
After predigestion 1h 430 DEG C digestion 3h, to digest tube in solution be changed into blue-tinted transparent；
3. room temperature is put in cooling, routinely protein assay method distills in kjeldahl apparatus and carries out result calculating, and protein changes
Calculate coefficient and take 5.79.
A kind of 6. method for differentiating oligopeptide albumen according to claim 5, it is characterised in that the step（1）- 2. in
DNA extract solutions are：The 10mmol/L Tris-Cl of pH=8.0, the 100mmol/L EDTA of pH=8.0, quality volume fraction 10%
SDS；Proteinase K is：200mg Proteinase K adds 9.5ml distilled waters, is settled to 20ml.
A kind of 7. method for differentiating oligopeptide albumen according to claim 5, it is characterised in that the step（1）- 8. in
TE solution is：The 10mmol/L Tris-Cl of pH=8.0, the 100mmol/L EDTA of pH=8.0.
A kind of 8. method for differentiating oligopeptide albumen according to claim 5, it is characterised in that the step（2）Middle PCR
Reaction system is：Containing Mg2+10 × PCR buffer 5 μ L, dNTP 4 μ L, 10 μm of ol/L upstream and downstream primer each 2 μ L, DNA
μ L, the 5U/ μ L of template 2 TaqE 0.6 μ L, ddH2O 34.4μL。
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| CN112210609A (en) * | 2020-10-20 | 2021-01-12 | 国家食品安全风险评估中心 | Real-time fluorescent PCR (polymerase chain reaction) primer, probe, kit and method for identifying puffer fish and application of real-time fluorescent PCR primer, probe, kit and method in grilled fish fillet |
| CN113061660A (en) * | 2020-12-25 | 2021-07-02 | 暨南大学 | Standard plasmid for identifying mandarin fish species and application thereof |
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| CN113061660A (en) * | 2020-12-25 | 2021-07-02 | 暨南大学 | Standard plasmid for identifying mandarin fish species and application thereof |
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Application publication date: 20180330 |