CN101712996A - Method for quickly identifying categories of meat and dried meat products of five domestic animals - Google Patents
Method for quickly identifying categories of meat and dried meat products of five domestic animals Download PDFInfo
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Abstract
The invention relates to a method for identifying categories of meat and dried meat products of five domestic animals (namely a pig, a goat, an ox, a buffalo and a yak), which is based on genetic variation of a 440bp gene segment of a chondriosome 12S rRNA gene, adopts a PCR-restriction fragment length polymorphism (PCR-RFLP) technique, and belongs to the field of biological high technology. The identification method of the invention comprises the following main steps: genomic DNA extraction of a sample, PCR amplification, restriction endonuclease digestion, endonuclease product detection and sample identification. The identification method of the invention has the characteristics of quickness, simpleness, economy and accuracy, which can be applied to massive detection of fresh meat and commercialized dried meat samples. The establishment of the method has important significance in the aspects of animal food inspection and quarantine, business fraud prevention and food backtracking system creation.
Description
Technical field:
The present invention relates to a kind of based on the segmental heritable variation of plastosome 12S rRNA gene 440bp, adopt PCR-restriction fragment length polymorphism (PCR-RFLP) technology, the method of Rapid identification pig, goat, ox, buffalo, 5 kinds of domestic animal meats of yak and dried meat product kind belongs to biological high-tech field.In the animal-derived food product inspection and quarantine, prevent commercial fraud, set up food and recall and trace system aspects and have great importance.
Background technology:
Along with the raising day by day of living standards of the people, the quantum of output of China's meat-based food and consumption all present the trend that increases year by year.Simultaneously, because the increase of consuming capacity and the change of consumption habit, to the also increase greatly of demand of the green meat-based food of health.And corresponding be that China's meat-based food processing and supervision link level seriously fall behind, the consumption market is lack of standardization, raw meat is mingled with shoddy commercial fraud behavior and is happened occasionally in the food.In addition, prevent that global animal epidemic from importing China into by the food import approach and also having become food test quarantine relevant departments and press for the problem of solution, as prevent that mad cow disease from importing China into by imported beef.China is a multi-nationality country, and each religion has different meat choice criteria, so the behavior of mingling in the meat-based food also will cause the religion culture and have a deep effect on.Therefore, set up a kind of fast, simple, economical, accurately livestock meat (comprising dried meat product) kind authentication method safeguard publilc health, prevent commercial fraud, all significant aspect the protection national belief.
RFLP is one of dna fingerprint technology that occurs the earliest, and its principle is to detect the specific DNA fragments electrophoretogram that DNA forms behind digestion with restriction enzyme.Different restriction enzymes can discern and the cutting DNA molecule in specific site, in case the base sequence in these sites changes then no longer discerned by its corresponding enzyme, non-restriction enzyme site also can produce a new recognition site owing to the sudden change of base simultaneously.Therefore, the every sudden change of restriction enzyme site change and enzyme that the general dna fragment deletion is caused of causing cut the change of product clip size, all can be used as the mark that species detect.The genetic marker that is usually used in the evaluation of domestic animal meat kind is mainly derived from Mitochondrial Genome Overview, comprises cytochrome b gene, control region sequence, 16S rRNA gene and 12S rRNA gene.Wherein, 12S rRNA gene is owing to have higher conservative type, and rate of evolution is moderate, and a less gene fragment has just comprised between kind and the genetic information in planting simultaneously, is normal adopted genetic marker.
By literature search, do not see the open report identical with the inventive method.
Summary of the invention:
The object of the present invention is to provide a kind of fast, simple, economical, differentiate the method for 5 kinds of livestock meats and dried meat product kind accurately.
The present invention is based on the segmental heritable variation of plastosome 12S rRNA gene 440bp, adopt PCR-restriction fragment length polymorphism (PCR-RFLP) technology, pig, goat, ox, buffalo, 5 kinds of domestic animal meats of yak and dried meat product kind thereof are identified.
The inventive method is by the pig of increasing simultaneously, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene 440bp fragment, analyze this fragment between species and the nucleotide site polymorphism information in planting, selecting AluI and two kinds of restriction enzymes of BfaI that the PCR product is carried out enzyme cuts, the result produces the endonuclease bamhi electrophoretogram of species specificity, realizes the evaluation to 5 kinds of domestic animal meats and dried meat product kind.The concrete steps of the inventive method are as follows:
(1) meat and dried meat product extracting genome DNA: use phenol/chloroform method and Guanidinium hydrochloride cracking process to extract genomic dna in green meat and the dried meat product;
(2) genomic dna that extracts with step (1) is a template, carries out the PCR reaction,
PCR reacts primer: amplimer upstream and downstream sequence is seen sequence table SEQ ID No.1 and SEQ ID No.2 respectively;
PCR reaction system: totally be 50 μ L, comprise the 100ng genomic dna, 10mM Tris-HCl (pH 8.3), 2.5mM MgCl
2, 50mM KCl, 10pM upstream and downstream primer, 1U Taq polysaccharase;
The PCR response procedures: 94 ℃, pre-sex change 4min; 94 ℃ of sex change 50s, 62 ℃ of annealing 50s, 72 ℃ are extended 60s, repeat 30 circulations; 72 ℃ are extended 10min.
(3) the PCR product detects: get PCR product 2 μ L electrophoresis on 1.0% agarose, electrophoretic buffer is 1 * TBE, 150V constant voltage electrophoresis 5min, and bromine second pyridine dyeing 5 minutes, gel imaging system is observed down and is taken pictures.
(4) digestion with restriction enzyme: 10 μ L AluI restriction endonuclease digestion reaction systems comprise 1 μ L PCR reaction product, 3U AluI, 1 μ L enzyme cutting buffering liquid; 37 ℃ of enzymes were cut 6 hours.10 μ L BfaI restriction endonuclease digestion reaction systems comprise 1 μ L PCR reaction product, 3U BfaI, 1 μ L enzyme cutting buffering liquid; 37 ℃ of enzymes were cut 6 hours.
(5) enzyme is cut the product somatotype: cancellationization product 8 μ L electrophoresis on 10% polyacrylamide gel, and electrophoretic buffer is 1 * TBE, 120V constant voltage electrophoresis 60min, bromine second pyridine dyeing 10 minutes, gel imaging system is observed down and is taken pictures.
(6) sample survey: cut product fragment length polymorphism electrophoretogram according to enzyme, the standard of perfection of each sample is:
AluI restriction endonuclease digestion: pork and dried meat product thereof, 162bp and 278bp; Goral mutton and dried meat product thereof, 200bp and 240bp; Carnis Bovis seu Bubali and dried meat product thereof, 91bp and 349bp; Buffalo meat and dried meat product thereof, 440bp; Carnis Bovis grunniens and dried meat product thereof, 91bp and 349bp.
The digestion of BfaI restriction endonuclease: pork and dried meat product thereof, 30bp, 100bp and 310bp, wherein 30bp is fuzzyyer on 10% polyacrylamide gel; Goral mutton and dried meat product thereof, 130bp and 310bp; Carnis Bovis seu Bubali and dried meat product thereof, 48bp, 82bp and 310bp; Buffalo meat and dried meat product thereof, 35bp, 130bp and 275bp; Carnis Bovis grunniens and dried meat product thereof, 130bp and 310bp.
Method of the present invention has fast, simple, economy, characteristic of accurate, can successful implementation in the flesh tissue of trace and commercialization jerky, and be suitable in extensive pattern detection, applying.Being based upon the animal-derived food product inspection and quarantine, preventing commercial fraud of this method, setting up food, to recall the system several respects of tracing all significant.
Description of drawings:
Fig. 1: the electrophoretogram of the AluI restriction endonuclease digestion product of pig, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene PCR amplified fragments.Wherein swimming lane M is the nucleic acid molecular weight standard of 50bp, and swimming lane 1 is the positive control of not digested PCR product, and swimming lane 2-3 is the sample of pig, swimming lane 4-5 is the sample of goat, swimming lane 6-7 is the sample of ox, and swimming lane 8-9 is the sample of buffalo, and swimming lane 10-11 is the sample of yak.
Fig. 2: the electrophoretogram of the BfaI restriction endonuclease digestion product of pig, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene PCR amplified fragments.Wherein swimming lane M is the nucleic acid molecular weight standard of 50bp, and swimming lane 1 is the positive control of not digested PCR product, and swimming lane 2-3 is the sample of pig, swimming lane 4-5 is the sample of goat, swimming lane 6-7 is the sample of ox, and swimming lane 8-9 is the sample of buffalo, and swimming lane 10-11 is the sample of yak.
Specific embodiments:
Embodiment 1: to the evaluation of pig, goat, ox, buffalo, 5 kinds of domestic animal green meats of yak
1, genetic marker is selected and design of primers
According to lot of documents report result, selection wire plastochondria 12S rRNA gene is as the genetic marker of identifying pig, goat, ox, buffalo, 5 kinds of livestock meat kinds of yak.Primer versatility good (the primer nucleotide sequence is seen SEQ ID No.1 and SEQ ID No.2 in the sequence table) can be used to the different plant species that increases simultaneously, and the prediction expanding fragment length is 440bp.Primer is synthetic by the handsome Bioisystech Co., Ltd in Shanghai.
2, restriction enzyme design
According to the pig of reporting among the GenBank, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene order, use Primer 5.0 software search restriction enzyme enzyme recognition sites, final AluI and two kinds of restriction enzymes of BfaI selected carry out PCR-restriction fragment length polymorphism (PCR-RFLP) analysis.
3, sample collection
The green meat sample of the pig that collection is identified through appearance, goat, ox, buffalo, 5 kinds of domestic animals of yak, each species is gathered 2 individualities.
4, sample gene group DNA extraction
Use phenol/chloroform method to extract genomic dna in the green meat.
5, pcr amplification
PCR reaction system: totally be 50 μ L, comprise the 100ng genomic dna, 10mM Tris-HCl (pH 8.3), 2.5mM MgCl
2, 50mM KCl, 10pM upstream and downstream primer, 1U Taq polysaccharase;
The PCR response procedures: 94 ℃, pre-sex change 4min; 94 ℃ of sex change 50s, 62 ℃ of annealing 50s, 72 ℃ are extended 60s, repeat 30 circulations; 72 ℃ are extended 10min.
6, the PCR product detects
Get PCR product 2 μ L electrophoresis on 1.0% agarose, electrophoretic buffer is 1 * TBE, 150V constant voltage electrophoresis 5min, and bromine second pyridine dyeing 5 minutes, gel imaging system is observed down and is taken pictures.
7, AluI restriction endonuclease digestion
Reaction system: total system 10 μ L comprise 1 μ L PCR reaction product, 3U AluI, 1 μ L enzyme cutting buffering liquid.
Reaction conditions: 37 ℃ of enzymes were cut 6 hours.
8, BfaI restriction endonuclease digestion
Reaction system: total system 10 μ L comprise 1 μ L PCR reaction product, 3U BfaI, 1 μ L enzyme cutting buffering liquid.
Reaction conditions: 37 ℃ of enzymes were cut 6 hours.
9, enzyme is cut the product detection
10, endonuclease bamhi length polymorphism (RFLP) banding pattern
Fig. 1 is that pig, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene PCR amplified fragments AluI enzyme cut the product electrophoretogram, wherein swimming lane 2-3 is the sample (162bp and 278bp) of pig, swimming lane 4-5 is the sample (200bp and 240bp) of goat, swimming lane 6-7 is the sample (91bp and 349bp) of ox, swimming lane 8-9 is the sample (440bp) of buffalo, and swimming lane 10-11 is the sample (91bp and 349bp) of yak.
Fig. 2 is that pig, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene PCR amplified fragments BfaI enzyme cut the product electrophoretogram, wherein swimming lane 2-3 is the sample (30bp, 100bp and 310bp) of pig, swimming lane 4-5 is the sample (130bp and 310bp) of goat, swimming lane 6-7 is the sample (48bp, 82bp and 310bp) of ox, swimming lane 8-9 is the sample (35bp, 130bp and 275bp) of buffalo, and swimming lane 10-11 is the sample (130bp and 310bp) of yak.
Embodiment 2: to the evaluation of commercialization dried beef
The method steps that the commercialization dried beef is identified is substantially with embodiment 1.Difference is:
1, is labeled as totally 8 parts of the dried beef of yak or Carnis Bovis seu Bubali in supermarket buying, is divided into spiced flavor, barbecue flavor, curried flavor, fragrant pungent and sauce pot-stewed fowl type.
2, use the Guanidinium hydrochloride cracking process to extract genomic dna in the dried beef.
3, all sample standard deviations show as two enzymes and cut banding pattern (91bp and 349bp) when using the digestion of AluI restriction endonuclease, and wherein 4 samples show as two enzymes and cut banding pattern (130bp and 310bp) when using the digestion of BfaI restriction endonuclease, are Carnis Bovis grunniens; Remaining 4 samples show as three enzymes and cut banding pattern (48bp, 82bp and 310bp), are Carnis Bovis seu Bubali.
Sequence table
＜110〉Kunming Institute of Zoology, Chinese Academy of Sciences
＜120〉method of 5 kinds of livestock meats of a kind of quick discriminating and dried meat product kind
＜130〉specification sheets
<160>2
<170>PatentIn?version?3.3
<210>1
<211>26
<212>DNA
＜213〉synthetic
<220>
<221>primer_bind
<222>(1)..(26)
<400>1
caaactggga?ttagataccc?cactat 26
<210>2
<211>20
<212>DNA
＜213〉synthetic
<220>
<221>primer_bind
<222>(1)..(20)
<400>2
gagggtgacgggcggtgtgt 20
Claims (1)
1. method of differentiating fast 5 kinds of domestic animal meats and dried meat product kind, it is characterized in that the inventive method pass through to increase simultaneously pig, goat, ox, buffalo, 5 kinds of domestic animal plastosomes of yak 12S rRNA gene 440bp fragment, analyze this fragment between species and the nucleotide site polymorphism information in planting, selecting AluI and two kinds of restriction enzymes of BfaI that the PCR product is carried out enzyme cuts, the result produces the endonuclease bamhi electrophoretogram of species specificity, realizes the evaluation to 5 kinds of domestic animal meats and dried meat product kind; The concrete steps of the inventive method are as follows:
(1) use phenol/chloroform method and Guanidinium hydrochloride cracking process to extract genomic dna in green meat and the dried meat product;
(2) be template with the genomic dna that extracts in green meat and the dried meat product, adopt the PCR reaction primer of having reported to carry out the PCR reaction:
PCR reaction system: totally be 50 μ L, comprise the 100ng genomic dna, 10mM Tris-HCl (pH 8.3), 2.5mM MgCl
2, 50mM KCl, 10pM upstream and downstream primer, 1U Taq polysaccharase;
The PCR response procedures: 94 ℃, pre-sex change 4min; 94 ℃ of sex change 50s, 62 ℃ of annealing 50s, 72 ℃ are extended 60s, repeat 30 circulations; 72 ℃ are extended 10min;
(3) the PCR product detects: get PCR product 2 μ L electrophoresis on 1.0% agarose, electrophoretic buffer is 1 * TBE, 150V constant voltage electrophoresis 5min, and bromine second pyridine dyeing 5 minutes, gel imaging system is observed down and is taken pictures;
(4) digestion with restriction enzyme: 10 μ L AluI restriction endonuclease digestion reaction systems comprise 1 μ L PCR reaction product, 3U AluI, 1 μ L enzyme cutting buffering liquid; 37 ℃ of enzymes were cut 6 hours.10 μ L BfaI restriction endonuclease digestion reaction systems comprise 1 μ L PCR reaction product, 3U BfaI, 1 μ L enzyme cutting buffering liquid; 37 ℃ of enzymes were cut 6 hours;
(5) enzyme is cut the product somatotype: cancellationization product 8 μ L electrophoresis on 10% polyacrylamide gel, and electrophoretic buffer is 1 * TBE, 120V constant voltage electrophoresis 60min, bromine second pyridine dyeing 10 minutes, gel imaging system is observed down and is taken pictures;
(6) sample survey: cut product fragment length polymorphism electrophoretogram according to enzyme, the standard of perfection of each sample is:
AluI restriction endonuclease digestion: pork and dried meat product thereof, 162bp and 278bp; Goral mutton and dried meat product thereof, 200bp and 240bp; Carnis Bovis seu Bubali and dried meat product thereof, 91bp and 349bp; Buffalo meat and dried meat product thereof, 440bp; Carnis Bovis grunniens and dried meat product thereof, 91bp and 349bp;
The digestion of BfaI restriction endonuclease: pork and dried meat product thereof, 30bp, 100bp and 310bp, wherein 30bp is fuzzyyer on 10% polyacrylamide gel; Goral mutton and dried meat product thereof, 130bp and 310bp; Carnis Bovis seu Bubali and dried meat product thereof, 48bp, 82bp and 310bp; Buffalo meat and dried meat product thereof, 35bp, 130bp and 275bp; Carnis Bovis grunniens and dried meat product thereof, 130bp and 310bp.
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