CN103215365A - DNA (Deoxyribonucleic Acid) molecular identification method for Luchuan pigs and meat products thereof - Google Patents
DNA (Deoxyribonucleic Acid) molecular identification method for Luchuan pigs and meat products thereof Download PDFInfo
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Abstract
The invention relates to a DNA (Deoxyribonucleic Acid) molecular identification method for Luchuan pigs and meat products thereof. According to the method, primers used in ISSR-PCR (Inter-Simple Sequence Repeat-Polymerase Chain Reaction) are WM14, WM12 and WM29, and nucleotide sequences of the primers are 5'-CTCCTCCTCGC-3', 5'-CACCACCACGC-3' and 5'-CTCCTCCTCAT-3'; and finally, seven electrophoretograms of the Luchuan pigs and the meat products thereof are drawn, a DNA fingerprint atlas of the Luchuan pigs and the meat products thereof is established, and the electrophoretograms and the DNA fingerprint atlas can serve as a basis for the distinction and identification of the Luchuan pigs and the meat products thereof. The method has the advantages that a reliable DNA molecular identification technique is adopted, so that the differences among the Luchuan pigs, the meat products of the Luchuan pigs, Luchuan pig hybrids, meat products of the Luchuan pig hybrids, Bama miniature pigs, meat products of the Bama miniature pigs, Huanjiang miniature pigs and meat products of the Huanjiang miniature pigs are identified quickly and accurately, and the development of advantaged brand pigs, namely the Luchuan pigs, is promoted.
Description
Technical field
The present invention relates to a kind of method of utilizing dna molecular marker technical evaluation pig variety, specifically is the dna molecular authentication method of a kind of Luchuan pig and meat product thereof.
Background technology
The Luchuan pig is the good pig kind of China South China, because of gaining the name its Luchuan County that originates in the Guangxi province southeast the existing areas such as Guangxi Yulin, Wuzhou, Qiezhou that mainly are distributed in.The Luchuan pig is with Rongchang County pig, Taihu Lake pig, Min pig, Jinhua pig, Ningxiang pig, Huaihe River pig and eight eyebrow pigs and claim " China's eight big local good pig kinds ".The Luchuan pig belongs to small-sized lard type kind, and the thin meat of Yin Qipi is tender, crisp and oiliness, delicious flavour, nutritious and have long enjoyed a good reputation.Its livestock product can be processed into goods such as sausage, no skin five flower bacon, white cut pig's feet, roasted suckling pig with crisp skin, braised pork, are subjected to local human consumer's favor deeply.But low because of its feed rate, breeding cycle is long, culture industry and many Hybrids occur, as with the Luchuan pig being the assorted pig of binary of female parent and the ternary pig of mixing.Simultaneously, because the Luchuan pork price on the market is several times of common pork price, the bad phenomenon of mixing the spurious with the genuine has appearred.On the other hand, the fragrant pig of fragrant pig of crust horse and ring river also is the famous-brand and high-quality pig kind in Guangxi, and roasting fragrant pig made from its sucking pig and the delicacy of cured fragrant pork matter, fragrant and sweet good to eat, fresh and sweet giving off a strong fragrance are liked by local people deeply, and found a good sale in the domestic and international market, the situation that supply falls short of demand occurs.Some illegal retailer is used to produce roasting fragrant pig and cured fragrant pig with the fragrant sucking pig of fragrant sucking pig of Luchuan sucking pig personation crust horse and ring river, and product has lost the genuineness of authentic fragrant pig, shoddy situation occurs.Still do not have efficient ways at present genuine piece Luchuan pork is carried out false distinguishing.
Be to estimate fresh meat and meat product quality thereof traditionally, comprise lean ratio, tender degree, pH value, yellowish pink etc. by measuring hog on hook and meat feature.But because raising condition and pig individuality there are differences, in the mensuration process acquisition time and sampling point for test agent there are requirement, bring to testing staff's work and disturb and inconvenience, cause the achievement data instability that obtains unreliable.By to meat discover that the meat index does not have unified numerical range mostly, individual difference is also apparent in view, can not be as the foundation of identifying pig variety.
Dna molecular marker (DNA molecular marker) is based on a kind of genetic marker of the polymorphism of biomacromolecule, has directly reflected the dna level genetic diversity.This technology directly with the form performance of DNA, all can detect in each tissue and each etap of organism, is not subjected to the restriction of sampling point, age, growth conditions etc.Dna molecular marker can detect unlimited locus, and quantity spreads all over whole genome and polymorphism height.These advantages are laid a good foundation for the dna molecular marker technology has widely applicability, have developed multiple molecule authentication technique at present, comprise RFLP technology, RAPD technology, AFLP technology, SSR technology and ISSR technology etc.Wherein ISSR technology and round pcr are united to use and are had very high specificity and sensitivity and accurately fast, be widely used in food quick test field, as detect transgene component, animals and plants species composition, pathogenic micro-organism etc., tracing to the source at food relates to as differentiating that fields such as food crop, fruit variety, vegetable variety also have.Heaton analyzes (HEATON M P by 32 SNP marks to evaluation and the parent that U.S.'s beef has carried out kind, HARHAY G P, BENNETT G L, et al.Selection and use of SNP markers for animal identification and paternity analysis in U.S.beef cattle[J] .Mammalian Genome, 2002,13 (5): 272-281).The old winter is once on the basis of amplification plastosome 12S rDNA gene, success identified mixing beef and the goods thereof on the market, and with this method identified 24 brands the dried yak beef goods (the old winter. differentiate the ox kind Source Study [D] of mixing beef and goods based on the PCR-RFLP of plastosome 12S rRNA gene. Yaan: Sichuan Agricultural University, 2008).Application number is that 201210057485.9 patents of invention disclose the method for utilizing ISSR molecular marking technique evaluation Pennisetum herbage, discloses primer UBC815 and UBC835, has distinguished 7 parts of Pennisetum herbage materials quickly and accurately.But do not see the report of domestic and international application dna molecular technology differentiation evaluation Luchuan pig variety.Utilize the dna molecular marker technology to have advantages such as multidigit point property, high variability, simple and stable heredity and somatocyte stability, foundation can be used for the specific DNA finger printing of the Luchuan pig of false distinguishing, the dna molecular authenticate technology of exploitation Luchuan pig and meat product thereof, to pig market, standard Luchuan, guide the sound development of deep processing enterprise and safeguard that " Luchuan pig " brand image has great importance.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides the dna molecular authentication method of a kind of Luchuan pig and meat product thereof, can identify the difference between Luchuan pig, Luchuan pig Hybrid, the fragrant pig of crust horse, the fragrant pig in ring river and the meat product thereof fast and accurately.
The present invention solves the problems of the technologies described above by the following technical solutions: the dna molecular authentication method of a kind of Luchuan pig and meat product thereof may further comprise the steps:
Total DNA of a, the fresh meat of extraction and meat product thereof;
B, utilize the DNA of fresh meat of ISSR-PCR reaction amplification and meat product thereof;
The DNA electrophoretogram of c, the fresh meat of structure and meat product thereof;
D, evaluation Luchuan pig and meat product thereof.
The used primer of described ISSR-PCR is WM14, WM12 and WM29, and wherein nucleotide sequence is respectively: 5'-CTCCTCCTCGC-3'; 5'-CACCACCACGC-3'; 5'-CTCCTCCTCAT-3'.
ISSR-PCR reaction system and program among the described step b are: 25 μ L systems: masterplate DNA1 μ L, primer 2 μ L, 2 * Taq PCR Master Mix12.5 μ L, ddH
2O supplies 25 μ L, and wherein 2 * Taq PCR Master Mix has comprised Taq archaeal dna polymerase, dNTPs, MgCl
2, reaction buffer, PCR reaction toughener and optimize agent and stablizer, concentration is 2 *, and do not contain dyestuff; Amplification program: 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, 36 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate 30 times, and 72 ℃ are extended 5min again.
With 1.5% sepharose, 1 * TAE damping fluid is a medium electrophoresis detection amplification among the described step c, draws electrophoretogram according to electrophorogram.
Described Luchuan pig is meant the Luchuan County that originates in the southeast, Guangxi Zhuang Autonomous Region, now mainly is distributed in Guangxi Yulin, Wuzhou, Qinzhou Prefecture, belongs to small-sized lard type kind.
Described meat product is meant with the Luchuan pig to be the braised pork and the cured meat product of raw material processing and preparing.
The application of the dna molecular authentication method of described Luchuan pig and meat product thereof in identifying the fresh meat of Luchuan pig and meat product and fresh meat of other pig variety and meat product thereof.
Described other pig variety is assorted for long Lu Eryuan, the continent binary assorted, the DLY ternary assorted, the long white ternary in continent is assorted, imperial precious pig, the assorted pig of ternary, the fragrant pig of crust horse and ring river perfume pig.
The method of total DNA of fresh meat of the described extraction of step a and meat product thereof is the SDS method after improveing.Used the phenol of liquid nitrogen, Proteinase K, 25:24:1 in the former method when cold: chloroform: reagent such as primary isoamyl alcohol, KAC, and need 50 ℃～55 ℃ following incubation a few hours, complex operation is used duration; Directly at ground sample on ice, replaced steps such as adding liquid nitrogen and incubation, and used the chloroform of saturated phenol and 24:1 respectively: primary isoamyl alcohol carries out twice extracting, has saved the time, has simplified step, has improved efficient, has reduced extraction cost when improving postcooling.
The electrophoretogram of described structure is chosen the finger printing that ISSR-PCR amplified production polymorphism is good, Luchuan pig and meat product thereof are intuitively identified in band electrophoretogram conduct clearly.
Substantive distinguishing features and marked improvement that the present invention gives prominence to are:
Utilize the dna molecular technology that Luchuan pig and other pig varieties of Guangxi are carried out Molecular Identification, efficiently solve the false distinguishing difficult problem in pig product source, Luchuan.The dna molecular technological operation simply, quick and precisely, rich polymorphism, template requirement be few, the testing efficient and the accuracy that have improved quality inspection personnel.
Constructed Luchuan pig specific DNA finger printing very intuitively shows the gene difference of Luchuan pig and some pig varieties of Guangxi, standard the operation of Luchuan pig developing market and deep processing enterprise, also significant to safeguarding " Luchuan pig " brand image.
Description of drawings
The Luchuan pig that Fig. 1 primer WM14 amplifies and the electrophoretogram of the fresh meat of Hybrid thereof;
The Luchuan pig that Fig. 2 primer WM14 amplifies and the electrophoretogram of Hybrid bacon thereof;
The Luchuan pig that Fig. 3 primer WM14 amplifies and the electrophoretogram of Hybrid braised pork thereof;
Luchuan pig that Fig. 4 primer WM12 amplifies and the electrophoretogram that encircles the fresh meat of the fragrant pig in river;
Luchuan pig that Fig. 5 primer WM12 amplifies and the electrophoretogram that encircles the fragrant pig bacon in river;
Luchuan pig that Fig. 6 primer WM29 amplifies and the electrophoretogram that clings to the fresh meat of the fragrant pig of horse;
Luchuan pig that Fig. 7 primer WM29 amplifies and the electrophoretogram that clings to the fragrant pig braised pork of horse;
Fig. 8 is the electrophoretic figure that draw on the basis with Fig. 1;
Fig. 9 is the electrophoretic figure that draw on the basis with Fig. 4;
Figure 10 is the electrophoretic figure that draw on the basis with Fig. 7.
Embodiment
Below by specific embodiment technical scheme of the present invention is described further.This test ISSR-PCR reaction primer is the sequence of design voluntarily, and synthetic by the prompt base in the English Weihe River (Shanghai) trade Co., Ltd.
Identify and distinguish the assorted pig of Luchuan pig and meat product and binary thereof, the assorted pig of ternary and meat product thereof
Choosing needs to identify Luchuan pig, the assorted pig of binary, the ternary assorted fresh meat of pig, bacon and braised pork sample number part, and the SDS method of employing improvement is extracted the DNA of each sample respectively.
With WM14 is primer, through the ISSR-PCR reaction, each total DNA that organizes sample is increased.The ISSR-PCR reaction system is 25 μ L systems: masterplate DNA1 μ L, primer 2 μ L, 2 * Taq PCR Master Mix12.5 μ L, ddH
2O9.5 μ L, wherein 2 * Taq PCR Master Mix has comprised Taq archaeal dna polymerase, dNTPs, MgCl
2, reaction buffer, PCR reaction toughener and optimize agent and stablizer, concentration is 2 *, and do not contain dyestuff; Amplification program: 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, 36 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate 72 ℃ of total elongation 5min 30 times.Wherein Marker is 100bp Plus DNA ladder, and add-on is 5 μ L, and the sample add-on is 8 μ L.
Sepharose with 1.5%, 1 * TAE damping fluid is a medium electrophoresis detection amplification, electrophoretogram such as Fig. 1,2,3, with Fig. 1 is electrophoretic figure such as Fig. 8 that draw on the basis, shown in arrow among each figure, the hybridized pig kind all produces a band more clearly, and does not have this band in the pig of Luchuan, repeatedly the repeated authentication experiment all can prove, proves that primer WM14 can be used as the strong evidence of distinguishing Luchuan pig and other Hybrid.
Wherein 1-6 represents six parts of fresh meat samples of different Luchuans pig among Fig. 1,9-10 represents four parts of assorted fresh meat samples of pig of different long Lu Eryuan, 11-14 represents four parts of different continents binary fresh meat sample of pig of mixing, 15-18 represent a imperial precious pig, two parts of different DLYs assorted pigs of outer ternarys and a continent long white in the assorted fresh meat sample of pig of ternary.1-5 represents five parts of different Luchuans pig bacon samples among Fig. 2,6-9 represents four parts of assorted pig bacon samples of different long Lu Eryuan, 10-12 represents three parts of different continents binary pig bacon sample of mixing, and 13-15 represents an assorted pig of ternary especially, 1 part of precious pig of dragon and a continent long white in the bacon sample of the assorted pig of ternary.1-5 represents five parts of different Luchuans pig braised pork samples among Fig. 3, and 6-9 represents four parts of assorted pig braised pork samples of different long Lu Eryuan, and 10-12 represents three parts of different continents binary pig braised pork sample of mixing, and 13-14 represents a ternary pig of mixing especially, the bacon sample of 1 part of precious pig of dragon.
Adopt NTSYS-pc2.10e software to carry out cluster analysis to distinguishing amplified band, with the SimQual program original matrix is asked genetic similarity (Jaccard coefficient), referring to table 1, wherein L1～L6 represents the Luchuan pig; C1～assorted the pig of the long Lu Eryuan of C4 representative; D1～D4 represents the continent binary pig of mixing; L represents the Long Bao pig; The assorted pig of the outer ternary of W1-W2 representative; Ternary is assorted in the N representative.The result shows that the Jaccard coefficient is 0.88～1.00 between the pig of Luchuan, illustrates that Luchuan pig sample room sibship is nearer.Jaccard coefficient between the assorted pig of Luchuan pig and long Lu Eryuan is 0.63～0.75; Jaccard coefficient between the assorted pig of Luchuan pig and continent binary is 0.63～0.75; Jaccard coefficient between Luchuan pig and the imperial precious pig is 0.63～0.75; Jaccard coefficient between the assorted pig of Luchuan pig and outer ternary is 0.50～0.63; Between the assorted pig of Luchuan pig and interior ternary the Jaccard coefficient be 0.50～0.63, show that all otherness is bigger between Luchuan pig and other the assorted pigs, it is far away that sibship differs.The present invention can accurately distinguish and differentiate Luchuan pig and other assorted pig varieties.
Jaccard matrix of coefficients between table 1 Luchuan pig and its hybridized pig
Identify and distinguish fragrant fresh meat of pig of Luchuan pig and meat product and ring river thereof and meat product thereof
Choosing needs to identify Luchuan pig, the fragrant fresh meat of pig in ring river and bacon sample number part thereof, and the SDS method of employing improvement is extracted the DNA of each sample respectively.
With WM12 is primer, and ISSR-PCR increases with embodiment 1.
Sepharose with 1.5%, 1 * TAE damping fluid are medium electrophoresis detection amplification, draw electrophoretogram 4,5 according to electrophorogram, are electrophoretic figure such as the Fig. 9 that draw on the basis with Fig. 4.Shown in arrow among each figure, the fragrant pig sample in ring river all produces a band more clearly, and does not have this band in the pig of Luchuan, and repeatedly the repeated authentication experiment all can prove, proves that primer WM12 can be used as the strong evidence of distinguishing the fragrant pig variety of Luchuan pig and ring river.
Wherein 1-5 represents five parts of fresh meat samples of different Luchuans pig among Fig. 4, and 6-10 represents the fresh meat sample of the fragrant pig in five parts of different rings rivers.1-4 represents four parts of different Luchuans pig bacon samples among Fig. 5, and 5-8 represents four parts of different rings Jiang Laxiang pig samples.
Adopt NTSYS-pc2.10e software to carry out cluster analysis, with the SimQual program original matrix is asked genetic similarity (Jaccard coefficient), referring to table 2 to distinguishing amplified band.The result shows that the Jaccard coefficient is that 0.86～1.00 explanation Luchuan pig sample room sibship is nearer between the pig of Luchuan.Jaccard coefficient between fragrant pig in ring river and the Luchuan pig is 0.43～0.57, proves that otherness is bigger between the fragrant pig of Luchuan pig and ring river, and it is far away that sibship differs.The present invention can be used for accurately distinguishing discriminating Luchuan pig and the fragrant pig in ring river.
Jaccard matrix of coefficients between the fragrant pig of table 2 Luchuan pig and ring river
Identify and distinguish fragrant fresh meat of pig of Luchuan pig and meat product and crust horse thereof and meat product thereof
Choosing needs to identify Luchuan pig, the fragrant fresh meat of pig of crust horse and braised pork sample number part thereof, and the SDS method of employing improvement is extracted the DNA of each sample respectively.
With WM29 is primer, and ISSR-PCR increases with embodiment 1.
Sepharose with 1.5%, 1 * TAE damping fluid are medium electrophoresis detection amplification, draw electrophoretogram 6,7 according to electrophorogram, are electrophoretic figure such as the Figure 10 that draw on the basis with Fig. 7.Shown in arrow among each figure, the fragrant pig sample of crust horse all produces a band more clearly, and does not have this band in the pig of Luchuan, and repeatedly the repeated authentication experiment all can prove, proves that primer WM29 can be used as the strong evidence of distinguishing Luchuan pig and the fragrant pig variety of Ba Ma.
Wherein 1-5 represents five parts of fresh meat samples of different Luchuans pig among Fig. 6, and 6-8 represents three parts of fresh meat samples of the different fragrant pigs of crust horse.1-4 represents four parts of different Luchuans pig braised pork samples among Fig. 7, and 5-8 represents the four parts of fragrant pig braised pork of different crust horses samples.
Adopt NTSYS-pc2.10e software to carry out cluster analysis, with the SimQual program original matrix is asked genetic similarity (Jaccard coefficient), referring to table 3 to distinguishing amplified band.The result shows that the Jaccard coefficient is 1.00 between the pig of Luchuan, illustrates that Luchuan pig sample room sibship is very approaching.Jaccard coefficient between Luchuan pig and the fragrant pig of crust horse is 0.75, proves that otherness is bigger between Luchuan pig and the fragrant pig of crust horse, and it is far away that sibship differs.The present invention can be used for accurately distinguishing discriminating Luchuan pig and the fragrant pig of crust horse.
Jaccard matrix of coefficients between table 3 Luchuan pig and the fragrant pig of crust horse
Claims (8)
1. the dna molecular authentication method of Luchuan pig and meat product thereof is characterized in that, may further comprise the steps:
Total DNA of a, the fresh meat of extraction and meat product thereof;
B, utilize the DNA of fresh meat of ISSR-PCR reaction amplification and meat product thereof;
The DNA electrophoretogram of c, the fresh meat of structure and meat product thereof;
D, evaluation Luchuan pig and meat product thereof.
2. the dna molecular authentication method of Luchuan as claimed in claim 1 pig and meat product thereof is characterized in that, the used primer of described ISSR-PCR is WM14, WM12 and WM29, and wherein nucleotide sequence is respectively: 5'-CTCCTCCTCGC-3'; 5'-CACCACCACGC-3'; 5'-CTCCTCCTCAT-3'.
3. the dna molecular authentication method of Luchuan as claimed in claim 1 pig and meat product thereof, it is characterized in that ISSR-PCR reaction system and program among the described step b are: 25 μ L systems: masterplate DNA1 μ L, primer 2 μ L, 2 * Taq PCRMaster Mix12.5 μ L, ddH
2O supplies 25 μ L, and wherein 2 * Taq PCR Master Mix has comprised Taq archaeal dna polymerase, dNTPs, MgCl
2, reaction buffer, PCR reaction toughener and optimize agent and stablizer, concentration is 2 *, and do not contain dyestuff; Amplification program: 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, 36 ℃ of annealing 30s, 72 ℃ are extended 2min, circulate 30 times, and 72 ℃ are extended 5min again.
4. the dna molecular authentication method of Luchuan as claimed in claim 1 pig and meat product thereof is characterized in that, with 1.5% sepharose, 1 * TAE damping fluid is a medium electrophoresis detection amplification among the described step c, draws electrophoretogram according to electrophorogram.
5. the dna molecular authentication method of Luchuan as claimed in claim 1 pig and meat product thereof, it is characterized in that, described Luchuan pig is meant the Luchuan County that originates in the southeast, Guangxi Zhuang Autonomous Region, mainly is distributed in Guangxi Yulin, Wuzhou, Qinzhou Prefecture, belongs to small-sized lard type kind.
6. the dna molecular authentication method of Luchuan as claimed in claim 1 pig and meat product thereof is characterized in that, described meat product is meant with the Luchuan pig to be the braised pork and the cured meat product of raw material processing and preparing.
7. as the application in identifying the fresh meat of Luchuan pig and meat product and fresh meat of other pig variety and meat product thereof of the dna molecular authentication method of any described Luchuan pig of claim 1～7 and meat product thereof.
8. application as claimed in claim 7 is characterized in that, described other pig variety is for long Lu Eryuan mixes, the continent binary is assorted, the DLY ternary is assorted, the long white ternary in continent is assorted, imperial precious pig ternary is mixed pig, the fragrant pig of fragrant pig of crust horse and ring river.
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| CN104593519A (en) * | 2015-02-26 | 2015-05-06 | 中国农业科学院上海兽医研究所 | Special primer, method and kit for identifying Meishan pig or meat product thereof |
| CN105349626A (en) * | 2015-10-09 | 2016-02-24 | 青岛农业大学 | Identification method of Laiwu pig and its meat products |
| CN105506167A (en) * | 2016-02-25 | 2016-04-20 | 河南农业大学 | Method for synchronously detecting animal-origin ingredients in meat and meat products |
| CN107475246A (en) * | 2017-09-05 | 2017-12-15 | 广西大学 | A kind of Luchuan pig and its DNA molecular authentication method of cooked meat product |
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| CN105543406B (en) * | 2016-03-16 | 2018-08-07 | 四川大学 | A method of utilizing ISSR molecular markers for identification BaMa miniature pigs |
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| CN104593519A (en) * | 2015-02-26 | 2015-05-06 | 中国农业科学院上海兽医研究所 | Special primer, method and kit for identifying Meishan pig or meat product thereof |
| CN105349626A (en) * | 2015-10-09 | 2016-02-24 | 青岛农业大学 | Identification method of Laiwu pig and its meat products |
| CN105506167A (en) * | 2016-02-25 | 2016-04-20 | 河南农业大学 | Method for synchronously detecting animal-origin ingredients in meat and meat products |
| CN107475246A (en) * | 2017-09-05 | 2017-12-15 | 广西大学 | A kind of Luchuan pig and its DNA molecular authentication method of cooked meat product |
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