CN105779630A - Short-tandem-repeats (STR) primer combination for source-traced identification on pork product and detection kit - Google Patents
Short-tandem-repeats (STR) primer combination for source-traced identification on pork product and detection kit Download PDFInfo
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Abstract
The invention discloses a short-tandem-repeats (STR) primer combination for source-traced identification on a pork product and a detection kit. The invention provides a primer kit for identifying pork. The primer kit is (1) or (2), wherein the (1) consists of primer pairs Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355, Swr1941, Sw2406, S0097, Sw857, S0226, Sw72, Sw122, S0090, IGF1, Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA; the (2) consists of a primer group A, a primer group B and a primer group C. According to the method provided by the invention, the selected 23 STR markers have the advantages that the polymorphism is high, the markers are not in linkage, amplified fragments have polymorphism, and genotyping is facilitated; grouped multiplex PCR (Polymerase Chain Reaction) detection can be achieved, the cost is reduced, the time is shortened, and the efficiency is increased.
Description
Technical field
The present invention relates to detection field of tracing to the source, particularly relate to a kind of pork product trace to the source differentiate micro-satellite primers combination and detection kit.
Background technology
In recent years due to economic profit incentive, market often occurs the phenomenon utilizing low-value product to pretend to be high-value product, counterfeit brand, the counterfeit place of production, not only upset market order, invaded consumer rights, but also probably due to the problems such as allergy cause the health risk of consumer.Although, what current China had started to implement meat can tracing management and application, but it is main using electron ear tage as primary identity, investigation display, the probability that ear tag comes off is on average about 10%, the ear tag expulsion rate of indivedual raisers is up to 50%, and easily occurs that again label is counterfeit owing to intermediate links are many, thus cannot realize really can reviewing.Carry out differentiating and tracing to the source it is thus desirable to develop a kind of effective method to the Carnis Sus domestica source on market.
Micro-satellite (STR) is many as a kind of DNA marker quantity in genome, it is wide and uniform to be distributed, polymorphism information content is high, stable in properties simultaneously, no matter raw meat or prepared food are all easy to detection, compared with other molecular markers (such as AFLP, RFLP, RAPD etc.), Microsatellite DNA Analysis method is convenient, it is appropriate to Aulomatizeted Detect and exploitation test kit, thus is widely used in the genetic affinity research of pig.Present invention employs the discriminating of tracing to the source for pork product of micro-satellite technology.
Summary of the invention
It is an object of the present invention to provide the primer set of discriminating of tracing to the source for Carnis Sus domestica.
The invention provides primer set, for following 1) or 2):
1) it is made up 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 up of primer sets A, primer sets B and primer sets C；
Described primer sets A is made up of primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355 and Swr1941；
Described primer sets B is made up of primer pair Sw2406, S0097, Sw857, S0226, Sw72, Sw122, S0090 IGF1；
Described primer sets C is made up of primer pair Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA；
In described primer pair Sw632 single strand dna shown in sequence in sequence table 1 and sequence table, the single strand dna shown in sequence 2 forms；
In described primer pair S0155 single strand dna shown in sequence in sequence table 3 and sequence table, the single strand dna shown in sequence 4 forms；
In described primer pair Sw2406 single strand dna shown in sequence in sequence table 5 and sequence table, the single strand dna shown in sequence 6 forms；
In described primer pair Sw951 single strand dna shown in sequence in sequence table 7 and sequence table, the single strand dna shown in sequence 8 forms；
In described primer pair Sw936 single strand dna shown in sequence in sequence table 9 and sequence table, the single strand dna shown in sequence 10 forms；
In described primer pair S0005 single strand dna shown in sequence in sequence table 11 and sequence table, the single strand dna shown in sequence 12 forms；
In described primer pair S0097 single strand dna shown in sequence in sequence table 13 and sequence table, the single strand dna shown in sequence 14 forms；
In described primer pair Sw787 single strand dna shown in sequence in sequence table 15 and sequence table, the single strand dna shown in sequence 16 forms；
In described primer pair Sw830 single strand dna shown in sequence in sequence table 17 and sequence table, the single strand dna shown in sequence 18 forms；
In described primer pair Sw2525 single strand dna shown in sequence in sequence table 19 and sequence table, the single strand dna shown in sequence 20 forms；
In described primer pair Sw857 single strand dna shown in sequence in sequence table 21 and sequence table, the single strand dna shown in sequence 22 forms；
In described primer pair S0107 single strand dna shown in sequence in sequence table 23 and sequence table, the single strand dna shown in sequence 24 forms；
In described primer pair S0225 single strand dna shown in sequence in sequence table 25 and sequence table, the single strand dna shown in sequence 26 forms；
In described primer pair S0226 single strand dna shown in sequence in sequence table 27 and sequence table, the single strand dna shown in sequence 28 forms；
In described primer pair Sw72 single strand dna shown in sequence in sequence table 29 and sequence table, the single strand dna shown in sequence 30 forms；
In described primer pair Sw2448 single strand dna shown in sequence in sequence table 31 and sequence table, the single strand dna shown in sequence 32 forms；
In described primer pair S0355 single strand dna shown in sequence in sequence table 33 and sequence table, the single strand dna shown in sequence 34 forms；
In described primer pair Swr1941 single strand dna shown in sequence in sequence table 35 and sequence table, the single strand dna shown in sequence 36 forms；
In described primer pair Sw911 single strand dna shown in sequence in sequence table 37 and sequence table, the single strand dna shown in sequence 38 forms；
In described primer pair Sw122 single strand dna shown in sequence in sequence table 39 and sequence table, the single strand dna shown in sequence 40 forms；
In described primer pair S0090 single strand dna shown in sequence in sequence table 41 and sequence table, the single strand dna shown in sequence 42 forms；
In described primer pair IGF1 single strand dna shown in sequence in sequence table 43 and sequence table, the single strand dna shown in sequence 44 forms；
In described primer pair CGA single strand dna shown in sequence in sequence table 45 and sequence table, the single strand dna shown in sequence 46 forms.
In above-mentioned primer set, a prime end mark fluorescent gene in described primer pair.
In above-mentioned primer set, 5 ' end labelling FAM of a primer in described primer pair Sw632；
5 ' end labelling TET of a primer in described primer pair S0155；
5 ' end labelling FAM of a primer in described primer pair Sw936；
5 ' end labelling TET of a primer in described primer pair S0005；
5 ' end labelling TET of a primer in described primer pair Sw830；
5 ' end labelling TET of a primer in described primer pair S0225；
5 ' end labelling TET of a primer in described primer pair S0355；
5 ' end labelling FAM of a primer in described primer pair Swr1941；
5 ' end labelling HEX of a primer in described primer pair Sw2406；
5 ' end labelling FAM of a primer in described primer pair S0097；
5 ' end labelling FAM of a primer in described primer pair Sw857；
5 ' end labelling HEX of a primer in described primer pair S0226；
5 ' end labelling HEX of a primer in described primer pair Sw72；
5 ' end labelling FAM of a primer in described primer pair Sw122；
5 ' end labelling HEX of a primer in described primer pair S0090；
5 ' end labelling HEX of a primer in described primer pair IGF1；
5 ' end labelling FAM of a primer in described primer pair Sw951；
5 ' end labelling FAM of a primer in described primer pair Sw787；
5 ' end labelling TET of a primer in described primer pair Sw2525；
5 ' end labelling HEX of a primer in described primer pair S0107；
5 ' end labelling TET of a primer in described primer pair Sw2448；
5 ' end labelling FAM of a primer in described primer pair Sw911；
5 ' end labelling FAM of a primer in described primer pair CGA.
Another object of the present invention is to provide the PCR reagent of discriminating of tracing to the source for Carnis Sus domestica.
PCR reagent provided by the invention, for following 1) or 2):
1) 1 in above-mentioned primer is included)；
2) by including the PCR reagent A of above-mentioned primer sets A, including the PCR reagent B of above-mentioned primer sets B and include the PCR reagent C of above-mentioned primer sets C and form；
In described PCR reagent A, every primer in every primer, S0355 in every primer, S0225 in every primer, SW830 in every primer, S0005 in every primer, SW936 in every primer, S0155 in the primer pair SW632 in described primer sets A, SWr1941 every primer mol ratio be 1.5:2:2:2.5:2:2:2.5:2；
In described PCR reagent B, in the primer pair SW2406 in described primer sets B in every primer, S0097 in every primer, SW857 in every primer, S0226 in every primer, SW72 in every primer, SW122 in every primer, S0090 in every primer, IGF1 every primer mol ratio be 2.5:2.5:3:2:1.5:2:2:3；
In described PCR reagent C, in the primer pair SW951 in described primer sets C in every primer, SW787 in every primer, SW2525 in every primer, S0107 in every primer, SW2448 in every primer and SW911CGA the mol ratio of every primer be 3.5:2.5:3:1.5:2:3:2.5.
In above-mentioned PCR reagent, in described PCR reagent A, concentration 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, 0.2 μm of ol/L, 0.25 μm of ol/L, 0.2 μm of ol/L of every primer, SWr1941 every primer in every primer, S0355 in every primer, S0225 in every primer, SW830 in every primer, S0005 in every primer, SW936 in every primer, S0155 in the primer pair SW632 in described primer sets A；
In described PCR reagent B, the concentration of every primer 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 μm of ol/L, 0.2 μm of ol/L, 0.2 μm of ol/L, 0.3 μm of ol/L in every primer, IGF1 in every primer, S0090 in every primer, SW122 in every primer, SW72 in every primer, S0226 in every primer, SW857 in every primer, S0097 in the primer pair SW2406 in described primer sets B；
In described PCR reagent C, in the primer pair SW951 in described primer sets C in every primer, SW787 in every primer, SW2525 in every primer, S0107 in every primer, 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 μm of ol/L.
The 3rd purpose of the present invention is to provide the test kit of discriminating of tracing to the source for Carnis Sus domestica.
Test kit provided by the invention, including above-mentioned primer or above-mentioned PCR reagent.
Above-mentioned primer or above-mentioned PCR reagent or the application in identifying Carnis Sus domestica individuality of the above-mentioned test kit are also the scope of protection of the invention.
Above-mentioned primer or above-mentioned PCR reagent or the application in identifying Carnis Sus domestica kind of the above-mentioned test kit are also the scope of protection of the invention；
Described Carnis Sus domestica kind is specially Pig Beijing Black, Min pig, duroc, ternary pigs or Taihu pigs.
Above-mentioned primer or above-mentioned PCR reagent or the application in pork product is traced to the source of the above-mentioned test kit are also the scope of protection of the invention.
The 4th purpose of the present invention is to provide a kind of method identifying Carnis Sus domestica kind to be measured.
Method provided by the invention, comprises the steps: with 2 in above-mentioned primer) shown in 3 primer sets respectively Carnis Sus domestica to be measured is carried out pcr amplification, obtain 3 groups of PCR primer；Again by described 3 groups of PCR primer capillary electrophoresis detection, identify Carnis Sus domestica kind to be measured according to pcr amplification product.
Identify that according to pcr amplification product the PCR primer of pig that Carnis Sus domestica kind to be measured is the different cultivars in the primer set by cattle to be measured in the PCR primer of all primer pairs and embodiment 2 or 3 carries out statistical analysis, thus judging which kind Carnis Sus domestica to be measured belongs to.
Described Carnis Sus domestica 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 inventive method are selected through creative work for applicant, and preferably obtain after China's native country great amount of samples checking, its primer sequence can inquire in the FAO (Food and Agriculture Organization of the United Nation) (FAO) microsatellite marker combination (http://dad.fao.org) for Genetic Diversity in Animal research with ISAG (ISAG) combine recommendation and pertinent literature.It has not chain between polymorphism height, labelling, amplified fragments and has polymorphism, it is easy to gene type.
(2) the inventive method attempts being grouped and utilize different fluorescent dye to modify the primer sequence of different marker sites, it may be achieved is grouped multiplex PCR detection, saves cost, shortens the time, improves efficiency.
(3) present invention achieves the discriminating of the microsatellite marker individual traceability qualification for pork product and part kind, the deficiency that the traceability system of perfect further current single electron ear tage exists, it is ensured that the listing retrospective uniqueness of whole process of Carnis Sus domestica, accuracy.
(4) the further application of the present invention, for hit manufacturing and marketing the fake of common occurrence on market, counterfeit brand and many infringements such as the place of production, counterfeit famous-brand and high-quality goods provide one efficiently, discrimination method accurately.
Accompanying drawing explanation
Fig. 1 is the Individual identification utilizing 23 pairs of STR primers to carry out Carnis Sus domestica.
Fig. 2 is the Variety identification utilizing 23 pairs of STR primers to carry out Carnis Sus domestica.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Below in conjunction with example, the specific embodiment of the present invention is described in further detail.
Embodiment 1, for identifying primer pair and the test kit thereof of Carnis Sus domestica kind
One, for identifying the primer pair design synthesis of Carnis Sus domestica kind
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 composite primer group and the reaction system thereof of Carnis Sus domestica kind
For improving pcr amplification efficiency, through repetition test, obtaining 3 composite primer groups (table 2) by being rationally grouped by 23 pairs of primers, screening obtains PCR compositional system and the amplification condition (table 3) of the best, establishes the best complex amplification method of 23 marker sites.Make 23 labelling seats only can obtain all purpose fragments by 3 PCR reactions.
Table 2 is the composite primer group for identifying Carnis Sus domestica kind
Table 3 is PCR system key component and final concentration
Each reaction system also includes 10 × pcr amplification buffer (TakaraTaqHotStart) and ddH2O。
Three, for identifying the test kit of Carnis Sus domestica kind
Test kit for identifying Carnis Sus domestica kind includes 3 PCR system of 23 primer pairs of above-mentioned one or 3 primer sets of above-mentioned two or above-mentioned two.
Four, for identifying the detection method of Carnis Sus domestica kind
1, pork product genomic DNA is extracted；
2, respectively genomic DNA is carried out multiplexed PCR amplification by 3 PCR system of above-mentioned two, obtain 3 kinds of pcr amplification products.
Pcr amplification program is as shown in table 4 below:
Table 4 is pcr amplification program
3, interpretation of result
Fluorescent labeling capillary electrophoresis is adopted to detect above-mentioned 3 kinds of pcr amplification products, size according to pcr amplification product and the color of fluorescence, it is judged that micro-satellite amplification, in conjunction with the micro-satellite amplification of the known Carnis Sus domestica of this kind, differentiated by statistical analysis, carry out identification of species.
2,23 primer pairs of embodiment identify that Carnis Sus domestica is individual
1, extracting genome DNA
Gather following Carnis Sus domestica sample respectively, Min pig 8 (being numbered M1-M8), duroc 4 (being numbered D1-D4), good assorted pig 3 (being numbered L1-L3), ternary pigs 13 (being numbered S1-S13), Yimeng black pig 4 (being numbered Y1-Y4), Taihu pigs 13 (being numbered T1-T13), and Pig Beijing Black 25 (being numbered H1-H25), amount to 70 Carnis Sus domestica samples.
Extract the genomic DNA of 70 Carnis Sus domestica samples respectively.
2, multiplexed PCR amplification
Respectively using the genomic DNA of 70 Carnis Sus domestica samples as template, carrying out pcr amplification by 3 reaction systems of the table 3 in embodiment 1, amplification program is table 4 such as.
3, upper machine testing
3 kinds of pcr amplification products of all Carnis Sus domestica samples are all gone up machine testing, uses 3730XL sequenator to detect.In 96 orifice plates, every hole adds mark and Methanamide mixed liquor (0.5:8.5) 9 μ l, PCR primer 1.0 μ l in molecular weight；95 DEG C of degeneration 3min, upper machine testing.
4, Individual identification interpretation of result
The PCR primer of all Carnis Sus domestica samples is as shown in table 5.
By each primer amplification result of every pig is analyzed, it is possible to find that every pig has oneself specific AFLP system.
By SIMCA-P14 software, utilizing offset minimum binary (OPLS-DA) diagnostic method that these 70 Carnis Sus domestica samples are differentiated, result is Fig. 1 such as, Pig Beijing Black (Blue), Min pig (Yellow), duroc (Green), ternary pigs (Light blue), Taihu pigs (Purple), good assorted pig (Red) and the black pig in Yimeng (Black) shown in, it can be seen that all of Carnis Sus domestica sample (each point represents a Carnis Sus domestica sample) is all independently distributed in this spectrogram.
Therefore, this method is utilized can to identify the Carnis Sus domestica from Different Individual.
3,23 primer pairs of embodiment are for the discriminating of different cultivars Carnis Sus domestica
1, extracting genome DNA
Identical with the 1 of embodiment 2；
2, multiplexed PCR amplification
Identical with the 2 of embodiment 2；
3, upper machine testing
Identical with the 3 of embodiment 2；
4, interpretation of result
23 kinds of combination of primers amplifications that all 7 kinds amount to 70 pigs are carried out offset minimum binary (OPLS-DA) diagnostic method analysis.Result as shown in fig. 2, it can be seen that utilize 23 pairs of pig SSR primers can to Pig Beijing Black (Blue), Min pig (Yellow), duroc (Green), ternary pigs (Light blue), Taihu pigs (Purple) 5 boars carry out extraordinary Variety identification.
23 kinds of combination of primers hence with the present invention, it is also possible to part Carnis Sus domestica kind is differentiated.
4,23 primer pair application in pork product is traced to the source of embodiment
1, extracting genome DNA
Select certain barren sow plant, take 3 Sanguis sus domestica samples frozen, be designated as ZX respectively1、ZX2、ZX3.Simultaneously artificial three pigs of tracing record from plant to slaughterhouse finally to the overall process of the market of farm produce or supermarket.The last meat sample gathering these three pigs from the market of farm produce or supermarket, is designated as ZR respectively1、ZR2、ZR3。
Extract ZX1、ZX2、ZX3、ZR1、ZR2、ZR3Genomic DNA.
2, multiplexed PCR amplification
Identical with the 2 of embodiment 2；
3, upper machine testing
Identical with the 3 of embodiment 2；
4, interpretation of result
Result is in Table 6, and comparison finds, the meat sample obtaining 3 pigs from market and the blood sample retained at that time with 23, STR primer is expanded after result spectrogram be on all four.This illustrates that this combination of primers may be used for reviewing of the question of market, and certain premise is that plant to leave blood sample hair equal samples.
Table 6 is that 23 primer pairs are traced to the source the blood sample of three pigs and meat sample amplification
Claims (10)
1. for identifying the primer set of Carnis Sus domestica, for following 1) or 2):
1) it is made up 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 up of primer sets A, primer sets B and primer sets C；
Described primer sets A is made up of primer pair Sw632, S0155, Sw936, S0005, Sw830, S0225, S0355 and Swr1941；
Described primer sets B is made up of primer pair Sw2406, S0097, Sw857, S0226, Sw72, Sw122, S0090 IGF1；
Described primer sets C is made up of primer pair Sw951, Sw787, Sw2525, S0107, Sw2448, Sw911 and CGA；
In described primer pair Sw632 single strand dna shown in sequence in sequence table 1 and sequence table, the single strand dna shown in sequence 2 forms；
In described primer pair S0155 single strand dna shown in sequence in sequence table 3 and sequence table, the single strand dna shown in sequence 4 forms；
In described primer pair Sw2406 single strand dna shown in sequence in sequence table 5 and sequence table, the single strand dna shown in sequence 6 forms；
In described primer pair Sw951 single strand dna shown in sequence in sequence table 7 and sequence table, the single strand dna shown in sequence 8 forms；
In described primer pair Sw936 single strand dna shown in sequence in sequence table 9 and sequence table, the single strand dna shown in sequence 10 forms；
In described primer pair S0005 single strand dna shown in sequence in sequence table 11 and sequence table, the single strand dna shown in sequence 12 forms；
In described primer pair S0097 single strand dna shown in sequence in sequence table 13 and sequence table, the single strand dna shown in sequence 14 forms；
In described primer pair Sw787 single strand dna shown in sequence in sequence table 15 and sequence table, the single strand dna shown in sequence 16 forms；
In described primer pair Sw830 single strand dna shown in sequence in sequence table 17 and sequence table, the single strand dna shown in sequence 18 forms；
In described primer pair Sw2525 single strand dna shown in sequence in sequence table 19 and sequence table, the single strand dna shown in sequence 20 forms；
In described primer pair Sw857 single strand dna shown in sequence in sequence table 21 and sequence table, the single strand dna shown in sequence 22 forms；
In described primer pair S0107 single strand dna shown in sequence in sequence table 23 and sequence table, the single strand dna shown in sequence 24 forms；
In described primer pair S0225 single strand dna shown in sequence in sequence table 25 and sequence table, the single strand dna shown in sequence 26 forms；
In described primer pair S0226 single strand dna shown in sequence in sequence table 27 and sequence table, the single strand dna shown in sequence 28 forms；
In described primer pair Sw72 single strand dna shown in sequence in sequence table 29 and sequence table, the single strand dna shown in sequence 30 forms；
In described primer pair Sw2448 single strand dna shown in sequence in sequence table 31 and sequence table, the single strand dna shown in sequence 32 forms；
In described primer pair S0355 single strand dna shown in sequence in sequence table 33 and sequence table, the single strand dna shown in sequence 34 forms；
In described primer pair Swr1941 single strand dna shown in sequence in sequence table 35 and sequence table, the single strand dna shown in sequence 36 forms；
In described primer pair Sw911 single strand dna shown in sequence in sequence table 37 and sequence table, the single strand dna shown in sequence 38 forms；
In described primer pair Sw122 single strand dna shown in sequence in sequence table 39 and sequence table, the single strand dna shown in sequence 40 forms；
In described primer pair S0090 single strand dna shown in sequence in sequence table 41 and sequence table, the single strand dna shown in sequence 42 forms；
In described primer pair IGF1 single strand dna shown in sequence in sequence table 43 and sequence table, the single strand dna shown in sequence 44 forms；
In described primer pair CGA single strand dna shown in sequence in sequence table 45 and sequence table, the single strand dna shown in sequence 46 forms.
2. primer set according to claim 1, it is characterised in that: a prime end mark fluorescent gene in described each primer pair.
3. primer set according to claim 1 and 2, it is characterised in that:
5 ' end labelling FAM of a primer in described primer pair Sw632；
5 ' end labelling TET of a primer in described primer pair S0155；
5 ' end labelling FAM of a primer in described primer pair Sw936；
5 ' end labelling TET of a primer in described primer pair S0005；
5 ' end labelling TET of a primer in described primer pair Sw830；
5 ' end labelling TET of a primer in described primer pair S0225；
5 ' end labelling TET of a primer in described primer pair S0355；
5 ' end labelling FAM of a primer in described primer pair Swr1941；
5 ' end labelling HEX of a primer in described primer pair Sw2406；
5 ' end labelling FAM of a primer in described primer pair S0097；
5 ' end labelling FAM of a primer in described primer pair Sw857；
5 ' end labelling HEX of a primer in described primer pair S0226；
5 ' end labelling HEX of a primer in described primer pair Sw72；
5 ' end labelling FAM of a primer in described primer pair Sw122；
5 ' end labelling HEX of a primer in described primer pair S0090；
5 ' end labelling HEX of a primer in described primer pair IGF1；
5 ' end labelling FAM of a primer in described primer pair Sw951；
5 ' end labelling FAM of a primer in described primer pair Sw787；
5 ' end labelling TET of a primer in described primer pair Sw2525；
5 ' end labelling HEX of a primer in described primer pair S0107；
5 ' end labelling TET of a primer in described primer pair Sw2448；
5 ' end labelling FAM of a primer in described primer pair Sw911；
5 ' end labelling FAM of a primer in described primer pair CGA.
4. for identifying the PCR reagent of Carnis Sus domestica, for following 1) or 2):
1) 1 in claim 1-3 in arbitrary described primer is included)；
2) by include in claim 1-3 arbitrary in described primer sets A PCR reagent A, include in claim 1-3 arbitrary in the PCR reagent B of described primer sets B and include in claim 1-3 arbitrary in the PCR reagent C of described primer sets C form；
In described PCR reagent A, every primer in every primer, S0355 in every primer, S0225 in every primer, SW830 in every primer, S0005 in every primer, SW936 in every primer, S0155 in the primer pair SW632 in described primer sets A, SWr1941 every primer mol ratio be 1.5:2:2:2.5:2:2:2.5:2；
In described PCR reagent B, in the primer pair SW2406 in described primer sets B in every primer, S0097 in every primer, SW857 in every primer, S0226 in every primer, SW72 in every primer, SW122 in every primer, S0090 in every primer, IGF1 every primer mol ratio be 2.5:2.5:3:2:1.5:2:2:3；
In described PCR reagent C, in the primer pair SW951 in described primer sets C in every primer, SW787 in every primer, SW2525 in every primer, S0107 in every primer, SW2448 in every primer and SW911CGA the mol ratio of every primer be 3.5:2.5:3:1.5:2:3:2.5.
5. PCR reagent according to claim 4, it is characterised in that:
In described PCR reagent A, concentration 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, 0.2 μm of ol/L, 0.25 μm of ol/L, 0.2 μm of ol/L of every primer, SWr1941 every primer in every primer, S0355 in every primer, S0225 in every primer, SW830 in every primer, S0005 in every primer, SW936 in every primer, S0155 in the primer pair SW632 in described primer sets A；
In described PCR reagent B, the concentration of every primer 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 μm of ol/L, 0.2 μm of ol/L, 0.2 μm of ol/L, 0.3 μm of ol/L in every primer, IGF1 in every primer, S0090 in every primer, SW122 in every primer, SW72 in every primer, S0226 in every primer, SW857 in every primer, S0097 in the primer pair SW2406 in described primer sets B；
In described PCR reagent C, in the primer pair SW951 in described primer sets C in every primer, SW787 in every primer, SW2525 in every primer, S0107 in every primer, 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 μm of ol/L.
6. for identifying the test kit of Carnis Sus domestica, including the described primer in arbitrary in claim 1-3 or the PCR reagent described in claim 4 or 5.
7. the PCR reagent described in any one described primer or claim 4 or 5 or the test kit described in claim 6 application in identifying Carnis Sus domestica individuality in claim 1-3.
8. the PCR reagent described in any one described primer or claim 4 or 5 or the test kit described in claim 6 application in identifying Carnis Sus domestica kind in claim 1-3；
Described Carnis Sus domestica kind is specially Pig Beijing Black, Min pig, duroc, ternary pigs or Taihu pigs.
9. the PCR reagent described in any one described primer or claim 4 or 5 or the test kit described in claim 6 application in pork product is traced to the source in claim 1-3.
10. identify Carnis Sus domestica kind to be measured a method, comprise the steps: with in claim 1-3 arbitrary described in primer in 2) shown in 3 groups of primer sets respectively Carnis Sus domestica to be measured is carried out pcr amplification, obtain 3 groups of PCR primer；Again described 3 groups of PCR primer are carried out capillary electrophoresis detection, identify Carnis Sus domestica kind to be measured according to pcr amplification product.
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| CN107557453A (en) * | 2017-09-12 | 2018-01-09 | 苏州博尔达生物科技有限公司 | Primer, probe, kit and its application based on RPA technology for detection pig derived components |
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