CN105671192A - DNA bar code establishing method for pork tracing and tracing method - Google Patents
DNA bar code establishing method for pork tracing and tracing method Download PDFInfo
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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
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Abstract
The invention discloses a DNA bar code establishing method for pork tracing.The method comprises the steps of 1, extracting genome DNA in pig individual tissue; 2, conducting PCR amplification with the genome DNA as the template, wherein corresponding primers are adopted to amplify gene ADAMTS, DAZL, FBXO32, FUT1, MC4R, MyoG and NR4A1 by one SNP locus and amplify gene PSMB by 2 SNP loci to obtain different amplified fragments; 3, conducing RFLP analysis, wherein enzyme digestion reaction is conducted on the amplified fragments with corresponding restriction enzymes, then agarose gel electrophoresis detection is conducted, the genotype corresponding to each fragment is judged according to the electrophoresis result, and the genotypes are assembled into a DNA bar code corresponding to the pig individual.The DNA bar code has high polymorphism information content and can be used for Duroc, Landrace and Large White pork tracing.The invention further discloses a pork tracing method based on the DNA bar code.
Description
Technical field
The invention belongs to technical field of bioengineering, it is specifically related to a kind of DNA bar code preparation method of tracing to the source for pork and the pork source tracing method based on described DNA bar code.
Background technology
It is point to pig farm to obtain domestic animals source that meat is traced to the source, namely cultivating, butcher, process and the link such as sale can trace back to the source individual information of meat. The detection of tracing to the source of meat in protection and improves in meat brand process and can play an important role; tracing to the source of same meat can also effectively distinguish when food crisis and recall contaminated food products; people pay attention to food quality and food safety gradually in recent years, and some scholars have carried out correlative study in the meat kinds such as ox, sheep and pig are traced to the source.
Molecule is traced to the source, and being labeled as traces to the source provides one to facilitate method, owing to each animal individual all dna sequences has uniqueness and stability, can accurately identify animal individual by differentiating the DNA characteristics of animal individual. Molecule is traced to the source to mark and is often adopted SNP marker, SNP (single nucleotide polymorphism, singlenucleotidepolymorphisms) mainly refer in genomic dna a certain specific nucleotide position changes, transversion, the DNA sequence dna caused by change such as insertion or disappearance polymorphism. SNP marker comprises following feature: widely distributed in genome of SNP site, has abundant genetic diversity, and the variation of many SNP and biological character is associated; SNP is diallele in genome, and a SNP site can produce 3 kinds of genotype.
Cultivating field market pig, duroc is distributed in countries in the world, is one of main male parent varieties in China's cross combination, in order to produce commodity lean meat pig; Landrace originates in Denmark, is world-renowned lean meat species pig kind, is the main kind of cross-breeding; Large White originates in Britain, is lean meat species pig kind famous and distributed more widely in the world. And owing to Duroc, great Bai and landrace are through seed selection for many years, cause some SNP site that inclined state phenomenon occurs, namely three kinds of genotype that SNP site produces can only detect out a kind of and two kinds of genotype, and this kind occurs that the SNP site of inclined state can not be used for tracing to the source of pork.Therefore molecule trace to the source tag application on pork is traced to the source time, the selection of SNP site determines that molecule is traced to the source the key factor of accuracy, is also that molecule is traced to the source the difficult point of technology for practical application.
China's pork labeling technique of tracing to the source still stops and traces to the source in technology at traditional label, easily there is leak in traceability chain of tracing to the source, pig individuality butchered segmentation after trace to the source be difficult to continue tracking go down, and molecule is traced to the source, marking method also needs supplementary perfect, also have bigger gap from practical application, prior art also has no the technology that can be used for the establishment DNA bar code of the combination based on SNP site genotype that pork is traced to the source.
Summary of the invention
The present invention is directed to and prior art also not can be used for this problem of technology based on SNP site genotype combination establishment DNA bar code that pork traces to the source, object be to provide a kind of DNA bar code traced to the source for pork preparation method and based on the pork source tracing method of described DNA bar code.
The principle of tracing to the source of the present invention may be summarized to be: the genotype measuring a series of SNP site of each individuality of pig population first in advance, work out DNA bar code using this as the identity code of individuality and to associate with its source-information one_to_one corresponding, generate DNA bar code-source-information database. If hereafter running into pork when needing to trace to the source, the genomic dna that only need to extract pork tissue measure described in the genotype of a series of SNP site and work out DNA bar code, by individuality source-information can be obtained with the DNA bar code matching identification in described database.
For realizing the practicality traced to the source, DNA bar code should have the abundant polymorphism information of content to ensure the difference identifiability of each individuality, and this is determined by the polymorphism information content of each SNP site selected and their mutual independence, therefore the selection of SNP site and combination are very crucial.
The present invention adopts restriction fragment length polymorphism polymerase chain reaction technique, i.e. RFLP-PCR (restrictionfragmentlengthpolymorphism-polymerasechainrea ctionamplification) technology, detection Duroc, the ADAMTS of great Bai and landrace colony (containing thrombospondin die body go de-connect metalloprotease gene), DAZL (similar azoospermia gene), FBXO32 (ubiquitin ligase gene), FUT1 (α-(1, 2) trehalose transferring enzyme 1 gene), MC4R (melanocortin receptor-4 gene), MyoG (myogenin gene), the polymorphism of the SNP site of NR4A1 (nuclear receptor 4A1) and PSMB (20S proteasome gene beta subunit gene 10), and the heterozygosity of SNP site is carried out computation and analysis, filter out 9 SNP site that polymorphism information content is abundant and separate, wherein each SNP site can detect three kinds of genotype, this provides guarantee for presenting different genotype establishment DNA bar code according to SNP site, the DNA bar code for pork is traced to the source can be worked out by measuring the genotype of above-mentioned 9 SNP site.
The preparation method of the DNA bar code traced to the source for pork of the present invention comprises step:
1) genomic dna in pig individuality tissue is extracted;
2) it is that template carries out pcr amplification taking genomic dna, use respectively corresponding primer pair Gene A DAMTS, DAZL, FBXO32, FUT1, MC4R, MyoG, NR4A1 increase respectively 1 SNP site, increase 2 SNP site to gene PS MB, obtains different amplified fragments;
3) rflp analysis, amplified fragments use corresponding restriction enzyme carry out endonuclease reaction respectively, again with agarose gel electrophoresis detection, judge, according to electrophoresis result, the genotype that each fragment is corresponding, and it is assembled into corresponding DNA bar code individual with described pig.
When adopting in RFLP technology the polymorphism detecting SNP site, the selection of the design of primer and screening, restricted shearing enzyme is comparatively important, the step 2 of the better embodiment of the one of the present invention) in, the primer sequence that pcr amplification uses is respectively,
ADAMTS: upstream primer sequence 5 '-TGGGGAGATTGTTCCAGAAC-3 ', downstream primer sequence 5 '-CTGCAGAACGAAGAAGTAGCC-3 ';
DAZL: upstream primer sequence 5 '-CGACAGGAATGAGTTTAGCAA-3 ', downstream primer sequence 5 '-CCAAAATGGAAGGGAAAAGT-3 ';
FBXO32: upstream primer sequence 5 '-ATCCCTGAGTGGCATTGCCCAA-3 ', downstream primer sequence 5 '-TTCAGCTGCTGCTGCCAGTG-3 '-3 ';
FUT1: upstream primer sequence 5 '-CTTCAGCCAGGGCTCCTTTAAG-3 ', downstream primer sequence 5 '-CTTCCTGAACGTCTATCAAGACC-3 ';
MC4R: upstream primer sequence 5 '-ATGAACTCAACCCATCACC-3 ', downstream primer sequence 5 '-TTAATATCTGCTAGACAAATCACAG-3 ';
MyoG: upstream primer sequence 5 '-TCAGGAAGAACTGAAGGCTG-3 ', downstream primer sequence 5 '-GTTTCCTGGGGTGTTGC-3 ';
NR4A1: upstream primer sequence 5 '-TTCCTCTGGGTCACAACG-3 ', downstream primer sequence 5 '-CTCACAGAGTCAATGCCG-3 ';
The corresponding sequence of two SNP site of PSMB is respectively
PSMB (1FR): upstream primer sequence 5 '-AGTTAAGCTCAGTCGTGCAG-3 ', downstream primer sequence 5 '-TGAAGTGGATCTTTTCGCAG-3 ';
PSMB (2FR): upstream primer sequence 5 '-AGCTGCGAAAAGATCCACTT-3 ', downstream primer sequence 5 '-GGTCTTCTAGCACTGCCAGG-3 '.
Further, step 3) in, endonuclease reaction restriction enzyme used is respectively:
ADAMTS:Pvu II;
DAZL:Msp I;
FBXO32:SnaB I;
FUT1:Hha I;
MC4R:Cla I;
MyoG:Msp I;
NR4A1:Dde I;
PSMB (1FR): Eco81 I;
PSMB (2FR): Eco72 I.
Pcr amplification needs certain condition could increase successfully, the step 2 of the better embodiment of the one of the present invention) in, pcr amplification condition is: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 52～64 DEG C of annealing 30s, 72 DEG C extend 30s, totally 30～40 circulations; Last 72 DEG C extend 10min.
Further, the annealing temperature of each gene PCR amplification correspondence is:
The form that DNA bar code can adopt numeral and letter to combine is worked out: 9 SNP site represent with numeral 1,2,3,4,5,6,7,8,9,3 kinds of genotype aa of each SNP site, bb, ab called after A, B, C respectively. Such as Gene A DAMTS is numbered 1, and SNP site is designated as SNP1, and in DNA bar code, correspondence is numbered 1A, 1B or 1C; Gene DAZL is numbered 2, and SNP site is designated as SNP2, and in DNA bar code, correspondence is numbered 2A, 2B or 2C; Analogizing successively, concrete coding is named according to individual detected result, and 9 couple numeral corresponding for 9 SNP site genotype and letter are combined to form the DNA bar code comprising SNP site information and genotype information.
9 SNP site genotype are separate, may be combined with into 39=19683 kinds of genotype, therefore the preparation method of the present invention can be used for the digitized bar code establishment that 19683 pig individual identity DNA identify in theory, substantially can meet the individual recognition that Duroc, great Bai and landrace meat are raised in pig farm, can be used for the identification of duroc, landrace and Large White meat product and trace to the source.
In practical application, based on the source tracing method of described DNA bar code, step can be comprised:
1) adopt method according to claim 1 to measure the individual corresponding DNA bar code of each pig of establishment in the raising stage, the DNA bar code of each pig individuality and source-information (comprising pig variety, growth information and sibship etc.) one_to_one corresponding typing are formed database;
2) extract pork tissue to be traced to the source and adopt method according to claim 1 to measure DNA bar code, then with the DNA bar code matching identification in database, and then association obtains the source-information of pork to be traced to the source.
Described pig is Duroc, great Bai and landrace.
Described source-information comprises pig variety, overbit record, growth record, breeding record, Breeding notes etc. and other relevant informations.
The positive progressive effect of the present invention is:
The present invention adopts RFLP-PCR method Duroc, long white and Large White kind to be detected, utilize 9 SNP site genotype of ADAMTS, DAZL, FBXO32, FUT1, MC4R, MyoG, NR4A1 and PSMB gene to set up the abundant DNA bar code of polymorphism information content, such that it is able to pork production, butcher, process and the link such as sale carries out pork and traces to the source detection.
Accompanying drawing explanation
Fig. 1 is the example photo of the electrophoresis detection of amplified fragments.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
For the establishment of the DNA bar code that Duroc, long white and Large White pork are traced to the source, concrete steps are:
1, the genomic dna in pig individuality tissue is extracted
Extracting and extract test kit with AXYGEN tissue DNA, produce by healthy and free from worry life science company limited, product type is AP-MN-MS-GDNA-50G.
1. cut and get 1-20mg pig ear tissue block, move into after shredding in 2mL centrifuge tube and (add 400 μ L physiological saline or 350 μ LPBS and 0.9 μ LRNaseA in advance), tissue grinder instrument grinds, to extract DNA better.
2. 150 μ LBufferC-L and 20 μ L Proteinase K vortex oscillation 1min immediately are added so that it is mix. Of short duration centrifugal rear (can not be centrifugal, observe tube wall inorganization block), be placed in 56 DEG C of water-bath 10min by centrifuge tube.
3. adding 350 μ LBufferP-D, vortex oscillation 30s makes it mix, the centrifugal 10min of 12000r.
4. prepared by DNA pipe and is placed in 2mL centrifuge tube, by step 3. in mixed solution move to DNA and prepare in pipe, the centrifugal 1min of 12000r.
5. abandon filtrate, pipe is prepared by DNA and is put back in original 2mL centrifuge tube, add the 500 centrifugal 1min of μ LBufferW1,12000r.
6. abandon filtrate, pipe is prepared by DNA and is put back in original 2mL centrifuge tube, add the 700 centrifugal 1min of μ LBufferW2,12000r.
7. repeating step is 6..
8. DNA being prepared the 1.5mL centrifuge tube that pipe is placed in another cleaning, prepares pipe film central authorities at DNA and add 100-200 μ LEluent or deionized water (preheating places 15min for 65 DEG C), room temperature leaves standstill the centrifugal 1min of 1min, 12000r, eluted dna.
9. centrifugal gained elutriant adds in adsorption column again, and the centrifugal 2min of 12000r, can obtain high-quality genomic dna.
2, pcr amplification
Using the genomic dna that extracts as amplification template, it may also be useful to primer is carried out pcr amplification by 9 respectively, primer synthesizes (primer sequence is in table 1) by Shanghai Sheng Gong bio-engineering corporation.
PCR reaction system is: 2 × TaqPCRMasterMix (5 μ L), ddH2O (3.4 μ L), upstream primer (10pmol/ μ L, 0.4 μ L), downstream primer (10pmol/ μ L, 0.4 μ L), genomic dna (50ng/mL, 0.8 μ L).
Amplification program is: 94 DEG C of denaturation 5min → (94 DEG C of sex change 30s → annealing 30s → 72 DEG C extend 30s) are circulated 35 times → 72 DEG C and extend 10min, and concrete annealing temperature when different genes increases is as shown in table 1.
Concrete grammar for the pcr amplification of 9 SNP site also can reference [1]-[8].
Table 1 is for detecting primer and the restriction endonuclease of 9 SNP site of 8 genes
3, enzyme is tested and genotype judgement conscientiously
(1) first whether detection amplification is successful, amplified fragments carries out agarose gel electrophoresis detection respectively, takes pictures after electrophoresis, and the single bright expression of PCR band is increased successfully, can be used for follow-up enzyme and cuts test.
(2) adopting restriction enzyme as shown in table 1 to carry out enzyme the successful amplified fragments of amplification of different SNP site respectively and cut test, enzyme carries out agarose gel electrophoresis detection again after cutting, then judges each SNP site genotype according to detected result.
Judge SNP site genotype principle as: there are two kinds of different forms for SNP site, be labeled as a, b, because being diallele, so there are aa, ab, bb tri-kinds of genotype. Due to relative restriction enzyme, the enzyme of the amplified fragments of a, b two kinds of forms being cut result and there is difference, amplified fragments carries out after enzyme cuts, and fragment length there will be polymorphism. Wherein the amplified fragments of aa type is not sheared, and fragment length is constant, and electrophoresis detection display is still a light belt; And the amplified fragments of bb type is cut into the less fragment of two length due to the existence of restriction enzyme, electrophoresis detection is then shown as two light belts; The electrophoresis detection of the amplified fragments of the assorted ab type closed then is shown as three light belts. Fig. 1 is 4 examples of electrophoresis detection photo, respectively the corresponding endonuclease bamhi of corresponding 7 Different Individual, MyoG gene M SP I endonuclease bamhi that wherein 1A is; 1B is PMSB (1FR) gene Eco81 I endonuclease bamhi; 1C is MC4R gene C la I endonuclease bamhi; 1D is DAZL gene M SP I endonuclease bamhi.
(3) sequence verification of the individual PCR primer of different genotype
To individual each 5 the 50 big systems amplifications of μ L of different genotype, the purifying order-checking of object band Hou Songshenggong biotechnology company limited is had to verify through agarose gel electrophoresis detection.
4, the establishment of DNA bar code
The DNA bar code that genotype establishment according to 9 SNP site is individual.
The form that DNA bar code adopts numeral and letter to combine is worked out: 9 SNP site represent with numeral 1,2,3,4,5,6,7,8,9,3 kinds of genotype aa of each SNP site, bb, ab called after A, B, C respectively. Such as Gene A DAMTS is numbered 1, and SNP site is designated as SNP1, corresponding in DNA bar code is numbered 1A, 1B or 1C, and concrete coding is named according to individual detected result; Gene DAZL is numbered 2, and SNP site is designated as SNP2, and in DNA bar code, correspondence is numbered 2A, 2B or 2C; Analogize successively, 9 couple corresponding for 9 SNP site genotype numeral and letter be combined to form the DNA bar code comprising SNP site information and genotype information. As shown in table 2 is the DNA bar code worked out by the detected result of 10 pig individualities.
The DNA bar code that table 2 is prepared according to 3 pig kind detected results
In above-mentioned table 4,1,2,3,4 and No. 6 pig individuality is Large White; 5 and No. 7 pig individualities are duroc, occur that 8 SNP are identical in their barcode, and SNP9 detects out different genotype in site. 8,9 and No. 10 pig individualities are landrace, detect out different genotype in SNP4 and SNP8 site respectively. The DNA bar code that visible 1～No. 10 pig individuality is corresponding different respectively, the form of 9 Sites Combination can ensure the difference between the DNA bar code of Different Individual, thus realizes the mark identification of Different Individual.
5, gene frequency and heterozygosity statistical study
During for actual tracing to the source, DNA bar code need to possess abundant polymorphism information, it is thus desirable to the heterozygosity H of each SNP site is carried out statistical study, H value shows that more greatly polymorphism information content is more abundant, the SNP of H value more than 0.30 is considered as significant notation site, thus could be used for individual differentiation and mark.
The genotype of each SNP site of duroc (100), Large White (150) and landrace (80) three kinds of swinerys is detected, gene frequency P (a), the P (b) of gene a, b according to detected result difference each SNP site of statistical computation and heterozygosity H
P (a)=(2A+C)/2 (A+B+C)
P (b)=(2B+C)/2 (A+B+C)
H=1-∑ pi
In above-mentioned relation formula, A, B, C represent the pig individual amount with this genotype respectively, and pi is this allelotrope polymorphism information content (plolymorphisminformationcontent, PIC) in colony. Statistics is as shown in table 3.
Table 39 SNP is at the gene frequency shown by 3 kinds and heterozygosity
In different pig kind, the H value in ADAMTS, FBXO32, NR4A1 and PSMB (2FR) site is all higher than 0.30, DAZL, FUT1, MC4R, MyoG and PSMB (1FR) gene only part pig kind H value be less than 0.3, H value scope is 0.21～0.53, shown polymorphism information content enriches, and can be used for mark of tracing to the source.
In order to guarantee in different time points, 330 bulk variety pigs to be comprised Duroc 100 in addition in the operability traced to the source in mark, Large White 150 and landrace 80, being sampled by each individuality and detect gene frequency and heterozygosity, result is as shown in table 4. Result shows 9 SNP site H values and is all greater than 0.3, and wherein the H value of SNP1 is 0.452; The H value of SNP2 is 0.415; The H value of SNP3 is 0.519; The H value of SNP4 is 0.357; The H value of SNP5 is 0.404; The H value of SNP6 is 0.315; The H value of SNP7 is 0.541; The H value of SNP8 is 0.336; The H value of SNP9 is 0.345. From, heterozygosity, these SNP site meet the requirement of mark of tracing to the source.
The gene frequency that table 49 SNP site is shown in 330 bulk varieties and heterozygosity
Therefore, the pork that the different genotype establishment DNA bar code that these 8 gene amplifications of ADAMTS, DAZL, FBXO32, FUT1, MC4R, MyoG, NR4A1 and PSMB can be adopted to go out 9 SNP site generations carries out duroc, Large White and landrace is traced to the source.
In practical application, Swine Production enterprise can adopt aforesaid method step the detection that all pigs individuality carries out DNA bar code to be worked out in the cultivation stage; The relevant information of recording individual pig simultaneously, comprising pig variety, overbit record, growth record, breeding record, Breeding notes. And with establishment DNA bar code one_to_one corresponding typing Production database. When pork needs to trace to the source, then detect its DNA bar code according to aforesaid method, identification of then comparing with the DNA bar code in database, and then obtain the source-information of individual pig to be traced to the source.
Reference
[1] Xu Shanshan, Yu Lili, Lekai. the genetic effect analysis of pig ADAMTS-1 gene pairs reproductive trait. [J]. Agriculture of Anhui science, 2008,36 (24): 10374-10376.
[2] Zhang Yuhao. pig H2A.Z, DAZL gene identification and genetic effect analysis. Master's thesis, 2008.
[3] in crystalline substance. the gene clone of pig FBXO32 and its transcription factor family, location, SNP detection and the association analysis with the production traits. Master's thesis .2006.
[4] Zhao Jinfeng, Zhang Dongjie, Gu Xi, etc. pig GPX5 gene, FUT1 gene and NCOA1 gene polynorphisms are analyzed. and [J]. North China agronomy report, 2008,23 (4): 69-71.
[5] Wang Wentao, Yang Xiuqin, He Xinmiao, Liu Di. the PCR-RFLP of five kind pig MC4R genes analyzes, [J]. HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE, 2008 (10): 29-30.
[6]SoumillionA,ErkensJH,LenstraJA,RettenbergerG,tePasMF.Geneticvariationintheporcinemyogeningenelocus.MammalianGenome.1997,8(8):564-568.
[7] Liu Linqing. the separation of pig HSD17B1, NR4A1, ALAS1 gene, qualification and Preliminary Study of Their Function. Ph D dissertation, 2009.
[8] Wu Xiao. pig ubiquitin-proteasome pathway location, sequence and trait associations analysis. Ph D dissertation, 2006.
Claims (8)
1. the DNA bar code preparation method traced to the source for pork, it is characterised in that, it comprises step:
1) genomic dna in pig individuality tissue is extracted;
2) it is that template carries out pcr amplification taking genomic dna, use respectively corresponding primer pair Gene A DAMTS, DAZL, FBXO32, FUT1, MC4R, MyoG, NR4A1 increase respectively 1 SNP site, increase 2 SNP site to gene PS MB, obtains different amplified fragments;
3) rflp analysis, amplified fragments use corresponding restriction enzyme carry out endonuclease reaction respectively, again with agarose gel electrophoresis detection, judge, according to electrophoresis result, the genotype that each fragment is corresponding, and it is assembled into corresponding DNA bar code individual with described pig.
2. the method for claim 1, it is characterised in that, step 2) in, the primer sequence that pcr amplification uses is respectively,
ADAMTS: upstream primer sequence 5 '-TGGGGAGATTGTTCCAGAAC-3 ', downstream primer sequence 5 '-CTGCAGAACGAAGAAGTAGCC-3 ';
DAZL: upstream primer sequence 5 '-CGACAGGAATGAGTTTAGCAA-3 ', downstream primer sequence 5 '-CCAAAATGGAAGGGAAAAGT-3 ';
FBXO32: upstream primer sequence 5 '-ATCCCTGAGTGGCATTGCCCAA-3 ', downstream primer sequence 5 '-TTCAGCTGCTGCTGCCAGTG-3 '-3 ';
FUT1: upstream primer sequence 5 '-CTTCAGCCAGGGCTCCTTTAAG-3 ', downstream primer sequence 5 '-CTTCCTGAACGTCTATCAAGACC-3 ';
MC4R: upstream primer sequence 5 '-ATGAACTCAACCCATCACC-3 ', downstream primer sequence 5 '-TTAATATCTGCTAGACAAATCACAG-3 ';
MyoG: upstream primer sequence 5 '-TCAGGAAGAACTGAAGGCTG-3 ', downstream primer sequence 5 '-GTTTCCTGGGGTGTTGC-3 ';
NR4A1: upstream primer sequence 5 '-TTCCTCTGGGTCACAACG-3 ', downstream primer sequence 5 '-CTCACAGAGTCAATGCCG-3 ';
The corresponding primer sequence of two SNP site of PSMB is respectively
PSMB (1FR): upstream primer sequence 5 '-AGTTAAGCTCAGTCGTGCAG-3 ', downstream primer sequence 5 '-TGAAGTGGATCTTTTCGCAG-3 ';
PSMB (2FR): upstream primer sequence 5 '-AGCTGCGAAAAGATCCACTT-3 ', downstream primer sequence 5 '-GGTCTTCTAGCACTGCCAGG-3 '.
3. method as claimed in claim 2, it is characterised in that, step 3) in, endonuclease reaction restriction enzyme used is respectively:
ADAMTS:Pvu II;
DAZL:Msp I;
FBXO32:SnaB I;
FUT1:Hha I;
MC4R:Cla I;
MyoG:Msp I;
NR4A1:Dde I;
PSMB (1FR): Eco81 I;
PSMB (2FR): Eco72 I.
4. method as claimed in claim 3, it is characterised in that, step 2) in, pcr amplification condition is: 94 DEG C of denaturation 4min; 94 DEG C of sex change 30s, 52～64 DEG C of annealing 30s, 72 DEG C extend 30s, totally 30～40 circulations; Last 72 DEG C extend 10min.
5. method as claimed in claim 4, it is characterised in that, the annealing temperature of each gene PCR amplification correspondence is:
6. the method traced to the source based on the pork of DNA bar code, it is characterised in that, comprise step:
1) adopt the individual corresponding DNA bar code of each pig in method according to claim 1 establishment swinery in the raising stage, the DNA bar code of each pig individuality and source-information one_to_one corresponding typing are formed database;
2) extract pork tissue to be traced to the source and adopt method according to claim 1 establishment DNA bar code, then with the DNA bar code matching identification in described database, and then association obtains the source-information of pork to be traced to the source.
7. method as claimed in claim 6, it is characterised in that, described pig is Duroc, great Bai and landrace.
8. method as claimed in claim 6, it is characterised in that, described source-information comprises pig variety, overbit record, growth record, breeding record, Breeding notes.
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| CN106566887A (en) * | 2016-11-07 | 2017-04-19 | 广西柯新源原种猪有限责任公司 | Group of SNP sites used for identifying Canada Duroc and Taiwan Duroc |
| CN113344593A (en) * | 2021-05-31 | 2021-09-03 | 优合集团有限公司 | Meat product traceability management system based on DNA detection technology |
| CN113344593B (en) * | 2021-05-31 | 2022-04-26 | 优合集团有限公司 | Meat product traceability management system based on DNA detection technology |
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