CN105779630B - It is a kind of for pork product trace to the source identification micro-satellite primers combination and its detection kit - Google Patents
It is a kind of for pork product trace to the source identification micro-satellite primers combination and its detection kit Download PDFInfo
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Abstract
The invention discloses a kind of pork product trace to the source identification micro-satellite primers combination and its detection kit.The present invention provides for identifying the primer set of pork, for it is following 1) or 2): 1) be made of primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355, Swr1941, Sw2406, S0097, Sw857, S0226, Sw72, Sw122, S0090, IGF1, Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA；2) it is made of primer sets A, primer sets B and primer sets C；23 microsatellite markers chosen in the method for the present invention have polymorphism with not chain, amplified fragments between polymorphism height, label, are easy to Genotyping, it can be achieved that being grouped multiplex PCR detection, save the cost, shortening the time, improve efficiency.
Description
Technical field
The present invention relates to trace to the source detection field more particularly to a kind of pork product trace to the source identification micro-satellite primers combination and
Its detection kit.
Background technique
In recent years due to economic profit incentive, often occurs pretending to be high-value product using low-value product in the market, imitate
The phenomenon that emitting brand, the counterfeit place of production, has not only upset market order, has invaded consumers' rights and interests, but also may be because allergy
The problems such as lead to the health risk of consumer.Although China has come into effect the traceable management and application of meat at present,
Mainly using electron ear tage as primary identity, investigation display, probability that ear tag falls off is average 10% or so, individual raisers'
Ear tag expulsion rate is up to 50%, and since to be mostly easy to appear label again counterfeit for intermediate links, so that cannot achieve can really chase after
It traces back.Therefore it needs to develop a kind of effective method pork source in the market is identified and traced to the source.
Microsatellite (STR) is more as a kind of DNA marker quantity in genome, distribution is wide and uniform, polymorphism information content
Height, while property is stablized, no matter raw meat or prepared food are all easy to detect, with other molecular labelings (such as AFLP, RFLP, RAPD
Deng) compare, Microsatellite DNA Analysis method is convenient, it is appropriate for automatic detection and exploitation kit, thus it is widely used in pig
Genetic affinity research in.Present invention employs the identifications of tracing to the source that microsatellite technology is used for pork product.
Summary of the invention
It is an object of the present invention to provide trace to the source the primer set of identification for pork.
The present invention provides primer set, for it is following 1) or 2):
1) by primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355, Swr1941, Sw2406,
S0097、Sw857、S0226、Sw72、Sw122、S0090、IGF1、Sw951、Sw787、Sw2525、S0107、Sw2448、Sw911
It is formed with CGA；
2) it is made of primer sets A, primer sets B and primer sets C；
The primer sets A is by primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355 and Swr1941
Composition；
The primer sets B is by Sw2406, S0097, Sw857, S0226, Sw72, Sw122, S0090 IGF1 groups of primer pair
At；
The primer sets C is made of primer pair Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA；
In primer pair Sw632 single strand dna as shown in sequence 1 in sequence table and sequence table shown in sequence 2
Single strand dna composition；
In primer pair S0155 single strand dna as shown in sequence 3 in sequence table and sequence table shown in sequence 4
Single strand dna composition；
In primer pair Sw2406 single strand dna as shown in sequence 5 in sequence table and sequence table shown in sequence 6
Single strand dna composition；
In primer pair Sw951 single strand dna as shown in sequence 7 in sequence table and sequence table shown in sequence 8
Single strand dna composition；
In primer pair Sw936 single strand dna as shown in sequence 9 in sequence table and sequence table shown in sequence 10
Single strand dna composition；
In primer pair S0005 single strand dna as shown in sequence 11 in sequence table and sequence table shown in sequence 12
Single strand dna composition；
In primer pair S0097 single strand dna as shown in sequence 13 in sequence table and sequence table shown in sequence 14
Single strand dna composition；
In primer pair Sw787 single strand dna as shown in sequence 15 in sequence table and sequence table shown in sequence 16
Single strand dna composition；
In primer pair Sw830 single strand dna as shown in sequence 17 in sequence table and sequence table shown in sequence 18
Single strand dna composition；
In primer pair Sw2525 single strand dna as shown in sequence 19 in sequence table and sequence table shown in sequence 20
Single strand dna composition；
In primer pair Sw857 single strand dna as shown in sequence 21 in sequence table and sequence table shown in sequence 22
Single strand dna composition；
In primer pair S0107 single strand dna as shown in sequence 23 in sequence table and sequence table shown in sequence 24
Single strand dna composition；
In primer pair S0225 single strand dna as shown in sequence 25 in sequence table and sequence table shown in sequence 26
Single strand dna composition；
In primer pair S0226 single strand dna as shown in sequence 27 in sequence table and sequence table shown in sequence 28
Single strand dna composition；
In primer pair Sw72 single strand dna as shown in sequence 29 in sequence table and sequence table shown in sequence 30
Single strand dna composition；
In primer pair Sw2448 single strand dna as shown in sequence 31 in sequence table and sequence table shown in sequence 32
Single strand dna composition；
In primer pair S0355 single strand dna as shown in sequence 33 in sequence table and sequence table shown in sequence 34
Single strand dna composition；
36 institute of sequence in primer pair Swr1941 single strand dna as shown in sequence 35 in sequence table and sequence table
The single strand dna composition shown；
In primer pair Sw911 single strand dna as shown in sequence 37 in sequence table and sequence table shown in sequence 38
Single strand dna composition；
In primer pair Sw122 single strand dna as shown in sequence 39 in sequence table and sequence table shown in sequence 40
Single strand dna composition；
In primer pair S0090 single strand dna as shown in sequence 41 in sequence table and sequence table shown in sequence 42
Single strand dna composition；
In primer pair IGF1 single strand dna as shown in sequence 43 in sequence table and sequence table shown in sequence 44
Single strand dna composition；
In primer pair CGA single strand dna as shown in sequence 45 in sequence table and sequence table shown in sequence 46
Single strand dna composition.
In above-mentioned primer set, a prime end mark fluorescent gene in the primer pair.
In above-mentioned primer set, 5 ' end mark FAM of a primer in the primer pair Sw632；
5 ' end mark TET of a primer in the primer pair S0155；
5 ' end mark FAM of a primer in the primer pair Sw936；
5 ' end mark TET of a primer in the primer pair S0005；
5 ' end mark TET of a primer in the primer pair Sw830；
5 ' end mark TET of a primer in the primer pair S0225；
5 ' end mark TET of a primer in the primer pair S0355；
5 ' end mark FAM of a primer in the primer pair Swr1941；
5 ' end mark HEX of a primer in the primer pair Sw2406；
5 ' end mark FAM of a primer in the primer pair S0097；
5 ' end mark FAM of a primer in the primer pair Sw857；
5 ' end mark HEX of a primer in the primer pair S0226；
5 ' end mark HEX of a primer in the primer pair Sw72；
5 ' end mark FAM of a primer in the primer pair Sw122；
5 ' end mark HEX of a primer in the primer pair S0090；
5 ' end mark HEX of a primer in the primer pair IGF1；
5 ' end mark FAM of a primer in the primer pair Sw951；
5 ' end mark FAM of a primer in the primer pair Sw787；
5 ' end mark TET of a primer in the primer pair Sw2525；
5 ' end mark HEX of a primer in the primer pair S0107；
5 ' end mark TET of a primer in the primer pair Sw2448；
5 ' end mark FAM of a primer in the primer pair Sw911；
5 ' end mark FAM of a primer in the primer pair CGA.
Another object of the present invention is to provide traces to the source the PCR reagent of identification for pork.
PCR reagent provided by the invention, for it is following 1) or 2):
1) include in above-mentioned primer 1)；
2) by PCR reagent A, the PCR reagent B including above-mentioned primer sets B including above-mentioned primer sets A and including above-mentioned primer
The PCR reagent C composition of group C；
In the PCR reagent A, every primer in the primer pair SW632 in the primer sets A, every primer in S0155,
Every primer in SW936, every primer in S0005, every primer in SW830, every primer in S0225, in S0355 every draw
Object, every primer of SWr1941 molar ratio be 1.5:2:2:2.5:2:2:2.5:2；
In the PCR reagent B, every primer in the primer pair SW2406 in the primer sets B, every primer in S0097,
Every primer in SW857, every primer in S0226, every primer in SW72, every primer in SW122, in S0090 every draw
Every primer molar ratio is 2.5:2.5:3:2:1.5:2:2:3 in object, IGF1；
In the PCR reagent C, every primer in the primer pair SW951 in the primer sets C, every primer in SW787,
Every primer in SW2525, every primer in S0107, in SW2448 in every primer and SW911CGA every primer molar ratio
It is 3.5:2.5:3:1.5:2:3:2.5.
In above-mentioned PCR reagent, in the PCR reagent A, every primer in the primer pair SW632 in the primer sets A,
Every primer in S0155, every primer in SW936, every primer in S0005, every primer in SW830, in S0225 every draw
Every primer in object, S0355, every primer of SWr1941 concentration be respectively 0.15 μm of ol/L, 0.2 μm of ol/L, 0.2 μm of ol/L,
0.25μmol/L,0.2μmol/L,0.2μmol/L,0.25μmol/L,0.2μmol/L；
In the PCR reagent B, every primer in the primer pair SW2406 in the primer sets B, every primer in S0097,
Every primer in SW857, every primer in S0226, every primer in SW72, every primer in SW122, in S0090 every draw
The concentration of every primer is respectively 0.25 μm of ol/L, 0.25 μm of ol/L, 0.3 μm of ol/L, 0.2 μm of ol/L, 0.15 μ in object, IGF1
mol/L,0.2μmol/L,0.2μmol/L,0.3μmol/L；
In the PCR reagent C, every primer in the primer pair SW951 in the primer sets C, every primer in SW787,
Every primer in SW2525, every primer in S0107, in SW2448 in every primer and SW911CGA every primer concentration
Respectively 0.35 μm of ol/L, 0.25 μm of ol/L, 0.3 μm of ol/L, 0.15 μm of ol/L, 0.2 μm of ol/L, 0.3 μm of ol/L, 0.25 μ
mol/L。
Third purpose of the present invention is to provide traces to the source the kit of identification for pork.
Kit provided by the invention, including above-mentioned primer or above-mentioned PCR reagent.
The application of above-mentioned primer or above-mentioned PCR reagent or above-mentioned kit in identification pork individual is also this hair
The range of bright protection.
The application of above-mentioned primer or above-mentioned PCR reagent or above-mentioned kit in identification pork kind is also this hair
The range of bright protection；
The pork kind is specially Pig Beijing Black, Min pig, duroc, ternary pigs or Taihu pigs.
The application of above-mentioned primer or above-mentioned PCR reagent or above-mentioned kit in tracing to the source pork product is also this
Invent the range of protection.
4th purpose of the invention is to provide a kind of method for identifying pork kind to be measured.
Method provided by the invention includes the following steps: with 2 in above-mentioned primer) shown in 3 primer sets it is right respectively
Pork to be measured carries out PCR amplification, obtains 3 groups of PCR products；Again by 3 groups of PCR product capillary electrophoresis detections, expanded according to PCR
Increase production object and identifies pork kind to be measured.
Identify that pork kind to be measured is by the PCR of primer pairs all in the primer set of ox to be measured according to pcr amplification product
The PCR product of the pig of different cultivars in product and embodiment 2 or 3 carries out statistical analysis, to judge that pork to be measured belongs to
Which kind.
The pork kind is specially Pig Beijing Black, Min pig, duroc, ternary pigs or Taihu pigs.
Beneficial effects of the present invention:
(1) 23 microsatellite markers chosen in the method for the present invention are selected for applicant through creative work, and through China
It is preferably obtained after the verifying of native country great amount of samples, primer sequence can be in FAO (Food and Agriculture Organization of the United Nation) (FAO) and international animal science of heredity
The microsatellite marker for Genetic Diversity in Animal research of meeting (ISAG) combine recommendation combines (http://dad.fao.org)
And it is inquired in pertinent literature.Not chain, amplified fragments have polymorphism between its, label high with polymorphism, are easy to gene point
Type.
(2) primer sequence of different marker sites is grouped and different fluorescent dyes is utilized to modify by the method for the present invention trial,
Grouping multiplex PCR detection can be achieved, save the cost, shorten the time, improve efficiency.
(3) present invention realizes microsatellite marker for the individual traceability identification of pork product and the mirror of part kind
Not, deficiency existing for the further perfect traceability system of current single electron ear tage guarantees that the whole process of listing pork is traceable
Uniqueness, accuracy.
(4) further application of the invention, for hitting commonplace in the market manufacturing and marketing the fake, counterfeit brand and production
Many illegal activities such as ground, counterfeit famous-brand and high-quality goods provide a kind of efficient, accurate discrimination method.
Detailed description of the invention
Fig. 1 is the Individual identification that pork is carried out using 23 pairs of STR primers.
Fig. 2 is the Variety identification that pork is carried out using 23 pairs of STR primers.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Below with reference to example, specific embodiments of the present invention will be described in further detail.
Embodiment 1, the primer pair for identifying pork kind and its kit
One, for identifying that the primer pair of pork kind designs synthesis
Design synthesizes following 23 pig micro-satellite primers pair, and sequence and Primer are as shown in table 1:
Table 1 is 23 pig micro-satellite primers pair
Two, for identifying the composite primer group and its reaction system of pork kind
3 composite primers are obtained by being rationally grouped 23 pairs of primers by repetition test to improve PCR amplification efficiency
Group (table 2), screening obtain optimal PCR compositional system and amplification condition (table 3), establish the best complex of 23 marker sites
Amplification method.So that 23 label seats is only passed through 3 PCR reactions can be obtained all purposes segment.
Table 2 is the composite primer group for identifying pork kind
Table 3 is PCR system main component and final concentration
It further include 10 × PCR amplification buffer (Takara Taq HotStart) and ddH in each reaction system2O。
Three, for identifying the kit of pork kind
For identify the kit of pork kind include above-mentioned one 23 primer pairs or above-mentioned two 3 primer sets or
State two 3 PCR systems.
Four, for identifying the detection method of pork kind
1, pork product genomic DNA is extracted；
2, multiplexed PCR amplification is carried out to genomic DNA respectively with above-mentioned two 3 PCR systems, obtains 3 kinds of PCR amplifications and produces
Object.
PCR amplification program is as shown in table 4 below:
Table 4 is PCR amplification program
3, interpretation of result
Using the above-mentioned 3 kinds of pcr amplification products of fluorescent marker capillary electrophoresis detection, according to the size of pcr amplification product and
The color of fluorescence judges microsatellite amplification, and pork microsatellite amplification, passes through statistical analysis in conjunction with known to the kind
Differentiate, carries out identification of species.
2,23 primer pair identification pork individuals of embodiment
1, extracting genome DNA
Following pork sample, 4 Min pig 8 (number M1-M8), duroc (number D1- are acquired respectively
D4), good miscellaneous pig 3 (number L1-L3), ternary pigs 13 (number S1-S13), the black pig 4 (number Y1-Y4) in Yimeng,
Taihu pigs 13 (number T1-T13) and Pig Beijing Black 25 (number is H 1-H25) amount to 70 pork samples.
The genomic DNA of 70 pork samples is extracted respectively.
2, multiplexed PCR amplification
Respectively using the genomic DNA of 70 pork samples as template, with 3 reaction systems of the table 3 in embodiment 1 into
Row PCR amplification, amplification program such as table 4.
3, upper machine testing
By the upper machine testing of 3 kinds of pcr amplification products of all pork samples, detected using 3730XL sequenator.?
Molecular weight internal standard and 9 μ l of formamide mixed liquor (0.5:8.5), 1.0 μ l of PCR product is added in every hole in 96 orifice plates；95 DEG C of denaturation
3min, upper machine testing.
4, Individual identification interpretation of result
The PCR product of all pork samples is as shown in table 5.
It is analyzed by each primer amplification result to every pig, oneself is specifically expanded it can be found that every pig has
Increase map.
By 14 software of SIMCA-P, this 70 pork samples are carried out using offset minimum binary (OPLS-DA) diagnostic method
Differentiate, as a result such as Fig. 1, Pig Beijing Black (Blue), Min pig (Yellow), duroc (It is green
Color), ternary pigs (It is light blue), Taihu pigs (Purple), good miscellaneous pig (It is red) and the black pig in Yimeng (Black) shown in, it can be seen that all pork samples (each select represents a pork sample) are independently distributed in this
In spectrogram.
Therefore, the pork from Different Individual can be identified using this method.
3,23 primer pairs of embodiment are used for the identification of different cultivars pork
1, extracting genome DNA
It is identical as the 1 of embodiment 2；
2, multiplexed PCR amplification
It is identical as the 2 of embodiment 2；
3, upper machine testing
It is identical as the 3 of embodiment 2；
4, interpretation of result
23 kinds of primers combination amplification that all 7 kinds amount to 70 pigs is subjected to offset minimum binary (OPLS-DA)
Diagnostic method analysis.As a result as shown in fig. 2, it can be seen that using 23 pairs of pig SSR primers can to Pig Beijing Black (It is blue
Color), Min pig (Yellow), duroc (Green), ternary pigs (It is light blue), Taihu pigs (Purple) the extraordinary Variety identification of 5 boars progress.
Therefore it is combined, part pork kind can also be identified using 23 kinds of primers of the invention.
4,23 applications of the primer pair in pork product is traced to the source of embodiment
1, extracting genome DNA
Certain pork pig farm is selected, takes 3 pig blood samples to freeze, is denoted as ZX respectively1、ZX2、ZX3.Artificial tracing record simultaneously
Three pigs are from farm to slaughterhouse finally to the market of farm produce or the overall process of supermarket.Finally this is acquired from the market of farm produce or supermarket
The meat sample of three pigs, is denoted as ZR respectively1、ZR2、ZR3。
Extract ZX1、ZX2、ZX3、ZR1、ZR2、ZR3Genomic DNA.
2, multiplexed PCR amplification
It is identical as the 2 of embodiment 2；
3, upper machine testing
It is identical as the 3 of embodiment 2；
4, interpretation of result
It the results are shown in Table 6, compare discovery, the meat sample and 23 pairs of STR primers of blood sample for retaining at that time of 3 pigs are obtained from market
Result spectrogram after being expanded is completely the same.This illustrates that the combination of this primer can be used for the retrospect of the question of market, certainly
On condition that farm will there are blood sample hair equal samples.
Table 6 is that 23 primer pairs are traced to the source the blood sample and meat sample amplification of three pigs
Claims (10)
1. for identifying the primer set of pork, for it is following 1) or 2):
1) by primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355, Swr1941, Sw2406, S0097,
Sw857, S0226, Sw72, Sw122, S0090, IGF1, Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA
Composition；
2) it is made of primer sets A, primer sets B and primer sets C；
The primer sets A is by primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355 and Swr1941 group
At；
The primer sets B is made of primer pair Sw2406, S0097, Sw857, S0226, Sw72, Sw122, S0090 and IGF1；
The primer sets C is made of primer pair Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA；
It is single-stranded shown in sequence 2 in primer pair Sw632 single strand dna as shown in sequence 1 in sequence table and sequence table
DNA molecular composition；
It is single-stranded shown in sequence 4 in primer pair S0155 single strand dna as shown in sequence 3 in sequence table and sequence table
DNA molecular composition；
It is single-stranded shown in sequence 6 in primer pair Sw2406 single strand dna as shown in sequence 5 in sequence table and sequence table
DNA molecular composition；
It is single-stranded shown in sequence 8 in primer pair Sw951 single strand dna as shown in sequence 7 in sequence table and sequence table
DNA molecular composition；
It is single-stranded shown in sequence 10 in primer pair Sw936 single strand dna as shown in sequence 9 in sequence table and sequence table
DNA molecular composition；
It is single shown in sequence 12 in primer pair S0005 single strand dna as shown in sequence 11 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 14 in primer pair S0097 single strand dna as shown in sequence 13 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 16 in primer pair Sw787 single strand dna as shown in sequence 15 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 18 in primer pair Sw830 single strand dna as shown in sequence 17 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 20 in primer pair Sw2525 single strand dna as shown in sequence 19 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 22 in primer pair Sw857 single strand dna as shown in sequence 21 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 24 in primer pair S0107 single strand dna as shown in sequence 23 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 26 in primer pair S0225 single strand dna as shown in sequence 25 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 28 in primer pair S0226 single strand dna as shown in sequence 27 in sequence table and sequence table
Ssdna molecule composition；
It is single-stranded shown in sequence 30 in primer pair Sw72 single strand dna as shown in sequence 29 in sequence table and sequence table
DNA molecular composition；
It is single shown in sequence 32 in primer pair Sw2448 single strand dna as shown in sequence 31 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 34 in primer pair S0355 single strand dna as shown in sequence 33 in sequence table and sequence table
Ssdna molecule composition；
In primer pair Swr1941 single strand dna as shown in sequence 35 in sequence table and sequence table shown in sequence 36
Single strand dna composition；
It is single shown in sequence 38 in primer pair Sw911 single strand dna as shown in sequence 37 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 40 in primer pair Sw122 single strand dna as shown in sequence 39 in sequence table and sequence table
Ssdna molecule composition；
It is single shown in sequence 42 in primer pair S0090 single strand dna as shown in sequence 41 in sequence table and sequence table
Ssdna molecule composition；
It is single-stranded shown in sequence 44 in primer pair IGF1 single strand dna as shown in sequence 43 in sequence table and sequence table
DNA molecular composition；
It is single-stranded shown in sequence 46 in primer pair CGA single strand dna as shown in sequence 45 in sequence table and sequence table
DNA molecular composition.
2. primer set according to claim 1, it is characterised in that: a prime end label is glimmering in each primer pair
Photogene.
3. primer set according to claim 1 or claim 2, it is characterised in that:
5 ' end mark FAM of a primer in the primer pair Sw632；
5 ' end mark TET of a primer in the primer pair S0155；
5 ' end mark FAM of a primer in the primer pair Sw936；
5 ' end mark TET of a primer in the primer pair S0005；
5 ' end mark TET of a primer in the primer pair Sw830；
5 ' end mark TET of a primer in the primer pair S0225；
5 ' end mark TET of a primer in the primer pair S0355；
5 ' end mark FAM of a primer in the primer pair Swr1941；
5 ' end mark HEX of a primer in the primer pair Sw2406；
5 ' end mark FAM of a primer in the primer pair S0097；
5 ' end mark FAM of a primer in the primer pair Sw857；
5 ' end mark HEX of a primer in the primer pair S0226；
5 ' end mark HEX of a primer in the primer pair Sw72；
5 ' end mark FAM of a primer in the primer pair Sw122；
5 ' end mark HEX of a primer in the primer pair S0090；
5 ' end mark HEX of a primer in the primer pair IGF1；
5 ' end mark FAM of a primer in the primer pair Sw951；
5 ' end mark FAM of a primer in the primer pair Sw787；
5 ' end mark TET of a primer in the primer pair Sw2525；
5 ' end mark HEX of a primer in the primer pair S0107；
5 ' end mark TET of a primer in the primer pair Sw2448；
5 ' end mark FAM of a primer in the primer pair Sw911；
5 ' end mark FAM of a primer in the primer pair CGA.
4. for identifying the PCR reagent of pork, for it is following 1) or 2):
1) include in any one of claim 1-3 primer set 1)；
2) by PCR reagent A including claim 1- including the primer sets A in any one of the claim 1-3 primer set
The PCR reagent B of primer sets B in 3 any one and including the primer sets C in claim any one of 1-3 PCR reagent C composition；
In the PCR reagent A, every primer in the primer pair SW632 in the primer sets A, every primer, SW936 in S0155
In every primer, every primer in S0005, every primer in SW830, every primer in S0225, every primer in S0355,
The molar ratio of every primer of SWr1941 is 1.5:2:2:2.5:2:2:2.5:2；
In the PCR reagent B, every primer in the primer pair SW2406 in the primer sets B, every primer in S0097,
Every primer in SW857, every primer in S0226, every primer in SW72, every primer in SW122, in S0090 every draw
Every primer molar ratio is 2.5:2.5:3:2:1.5:2:2:3 in object, IGF1；
In the PCR reagent C, every primer in the primer pair SW951 in the primer sets C, every primer in SW787,
Every primer in SW2525, every primer in S0107, every primer rubs in every primer and SW911 and CGA in SW2448
Your ratio is 3.5:2.5:3:1.5:2:3:2.5.
5. PCR reagent according to claim 4, it is characterised in that:
In the PCR reagent A, every primer in the primer pair SW632 in the primer sets A, every primer, SW936 in S0155
In every primer, every primer in S0005, every primer in SW830, every primer in S0225, every primer in S0355,
The concentration of every primer of SWr1941 is respectively 0.15 μm of ol/L, 0.2 μm of ol/L, 0.2 μm of ol/L, 0.25 μm of ol/L, 0.2 μm of ol/
L,0.2μmol/L,0.25μmol/L,0.2μmol/L；
In the PCR reagent B, every primer in the primer pair SW2406 in the primer sets B, every primer in S0097,
Every primer in SW857, every primer in S0226, every primer in SW72, every primer in SW122, in S0090 every draw
The concentration of every primer is respectively 0.25 μm of ol/L, 0.25 μm of ol/L, 0.3 μm of ol/L, 0.2 μm of ol/L, 0.15 μ in object, IGF1
mol/L,0.2μmol/L,0.2μmol/L,0.3μmol/L；
In the PCR reagent C, every primer in the primer pair SW951 in the primer sets C, every primer in SW787,
Every primer in SW2525, every primer in S0107, every primer in every primer and SW911 and CGA in SW2448
Concentration is respectively 0.35 μm of ol/L, 0.25 μm of ol/L, 0.3 μm of ol/L, 0.15 μm of ol/L, 0.2 μm of ol/L, 0.3 μm of ol/L, 0.25
μmol/L。
6. for identifying any one of kit, including claim 1-3 of pork primer set or claim 4 or 5 institutes
State PCR reagent.
7. being tried described in any one of the claim 1-3 primer set or the PCR reagent of claim 4 or 5 or claim 6
Application of the agent box in identification pork individual.
8. being tried described in any one of the claim 1-3 primer set or the PCR reagent of claim 4 or 5 or claim 6
Application of the agent box in identification pork kind；
The pork kind is specially Pig Beijing Black, Min pig, duroc, ternary pigs or Taihu pigs.
9. being tried described in any one of the claim 1-3 primer set or the PCR reagent of claim 4 or 5 or claim 6
Application of the agent box in tracing to the source pork product.
10. a kind of method for identifying pork kind to be measured, include the following steps: complete to be drawn with any one of claim 1-3 is described
2 in object) 3 groups of primer sets shown in carry out PCR amplification to pork to be measured respectively, obtain 3 groups of PCR products；Again by described 3 groups
PCR product carries out capillary electrophoresis detection, identifies pork kind to be measured according to pcr amplification product.
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| CN1659179A (en) * | 2002-04-08 | 2005-08-24 | 猪改良英国有限公司 | System for tracing animal products |
| CN102154280A (en) * | 2011-03-08 | 2011-08-17 | 公安部物证鉴定中心 | Method for multiplex amplification of pig microsatellite marker and special primer thereof |
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| CN1659179A (en) * | 2002-04-08 | 2005-08-24 | 猪改良英国有限公司 | System for tracing animal products |
| CN102154280A (en) * | 2011-03-08 | 2011-08-17 | 公安部物证鉴定中心 | Method for multiplex amplification of pig microsatellite marker and special primer thereof |
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| DRAFT GUIDELINES ON MOLECULAR GENETIC CHARACTERIZATION OF ANIMAL GENETIC RESOURCES;Anonymous;《COMMISSION ON GENETIC RESOURCES FOR FOOD AND AGRICULTURE（Thirteenth Regular Session）》;20110722;第63-65页 * |
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