KR101630806B1 - Discrimination method for product traceability and identification of pork using Microsatellite DNA - Google Patents

Abstract

The present invention relates to the development of supersatellite markers for the gene identification of pigs, and more particularly, to a more rapid and economical method than conventional techniques by multiplex PCR using microsatellite markers To identify the individual and to identify the parent. Multiplex polymerization reaction method of 23 microsatellite markers according to the present invention can be used very usefully in a pig hysteresis tracer by performing high speed and low cost analysis using a gene for identification of a pig and identification of a parent have.

Description

{Discrimination method for product traceability and identification of pork using microsatellite DNA}

The present invention relates to the development of supersatellite markers for the gene identification of pigs, and more particularly, to a more rapid and economical method than conventional techniques by multiplex PCR using microsatellite markers To identify the individual and to identify the parent.

In general, tracking the origin of livestock products (pork) is conducted in order to prevent malignant diseases such as money cholera and foot-and-mouth disease from spreading to people and animals and animals in other areas in advance.

To this end, in the case of Europe, only pigs that have been identified from their origin are obliged to be labeled (barcode label attached to their ears) so that they can be slaughtered or moved. After slaughtering, Adherence has led to a system of tracking the origin of pork at the point of sale.

However, in Japan and Korea, since the price difference between foreign pork and domestic pork is very high, there is a tendency that information of origin is intentionally blocked after slaughter. Even if it is not intentional, it is not linked to perfect origin information due to errors in the identification system (table) of traditional pigs. In some countries, it is attempting to verify the authenticity of origin information through the introduction of gene detection techniques. However, since the domains that can be identified on the pig's genome vary according to the variety of pigs, it is necessary to select the marker genes based on the specific hereditary pattern of the native pigs for identification of the native pigs, It is very important to set up gene detection techniques.

Meanwhile, in Korea, genetic diversity studies between breeders and genetic markers (microsatellite DNA) developed for paternity identification have been mostly used in conservation aspects of traditional pork genes. However, when the above-mentioned genetic markers are partially used for gene detection of native pigs, there is a disadvantage that their usefulness and accuracy are inferior. In addition, since some of the currently developed gene markers are used only for the purpose of paternity identification, more useful gene markers are selected to enhance the effectiveness of identification of native pigs and verification of origin, There is a need to develop a sufficient number of genetic markers to be identified.

Korean Patent Publication No. 10-2005-0119274

In order to solve the conventional problems as described above, the inventors of the present invention have found that, in order to solve the above-mentioned conventional problems, the inventors of the present invention have found that, in comparison with the gene genetic identification information analyzed from samples collected after being slaughtered and transported or sold to a retailer, We selected a variety of porcine genetic markers that could be used to quickly and accurately verify identification and paternity identification, and developed an analysis method using the genetic markers.

Accordingly, an object of the present invention is to select a gene marker from a porcine super-satiety gene and to use it to track the origin of pork and to identify the individual according to the characteristics of the breed, which is excellent in quickness and accuracy.

According to one aspect of the present invention, there is provided a method for amplifying supernatant DNA to identify an individual of pigs, comprising the steps of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, may be provided.

According to another aspect of the present invention, there is provided a kit for identifying a pig, comprising a combination of the primer pairs.

According to still another aspect of the present invention, there is provided a method of detecting DNA comprising: extracting DNA from a sample; Amplifying the extracted DNA by a polymerase chain reaction with the primer pair of claim 1; And analyzing the genotype of the product of the PCR reaction can be provided.

In one embodiment, the polymerase chain reaction is carried out at 95 ° C for 15 minutes, followed by reaction at 94 ° C for 40 seconds, 59 to 60 ° C for 35 to 45 seconds, and 72 ° C for 40 seconds. , And allowed to react at 72 ° C for 10 minutes.

According to one embodiment, the porcine supernatant DNA has 3 to 6 alleles, a heterozygote occurrence rate of 60% or higher, and a gene size of 81 to 289 bp.

Multiplex polymerization reaction method of 23 microsatellite markers according to the present invention can be used very usefully in a pig hysteresis tracer by performing high speed and low cost analysis using a gene for identification of a pig and identification of a parent have.

In order to achieve the above object, the present invention provides a method for amplifying a supersatellite gene in a specimen using a gene amplification primer specific to a super-satiety locus of a pig, separating the amplified gene product by size, Wherein the size of the amplified gene product is measured and then compared with the control group.

Hereinafter, the present invention will be described in detail.

Here, unless otherwise defined in the technical terms and the scientific terms used, those having ordinary skill in the art to which the present invention belongs have the same meaning as commonly understood by those skilled in the art.

In addition, repeated description of the same technical structure and operation as the conventional one will be omitted.

The present invention uses a microsatellite DNA that is separated from a main chromosome by high-level centrifugation in a chromosome, as a gene marker for individual identification in blood and other tissue samples of native pigs.

The animals and DNA samples used in the present invention are five pigs, consisting of Korean native pigs, Berkshire, Landrace, Yorkshire and Duroc, Genomic DNA was isolated from the blood using a genomic DNA extraction kit (Promega, USA), using 96 clones, 19 clones each, excluding the two clones.

The present invention relates to a method for gene detection by a multiplex PCR method for identification of a gene in a porcine. The selected microsatellite markers include a microsatellite marker, a Mapviewer database of National Center for Biotechnology Information (NCBI) And allele frequency of the allele genotype, annealing temp. Of the primer, product size of the amplified product, fluorescent substance (Dye), and finally divided into 23 sets so that multiplex PCR can be performed.

At this time, the gene diagnostic primer can prepare two kinds of primers per genetic label to analyze the alleles, and perform PCR amplification reaction together with the porcine satellites DNA to detect each gene marker specifically expressed in pigs Star allele (Allele) is amplified.

The amplified alleles are classified into the number of alleles, the position of the gene, the gene size, and the heterozygote occurrence rate for each gene marker using a suitable analyzer.

Then, the present invention secures a highly effective gene marker that appears at a high frequency in the pig population from the selection method described above.

According to one aspect of the present invention, there is provided a method for amplifying supernatant DNA to identify an individual of pigs, comprising the steps of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, may be provided.

According to another aspect of the present invention, there is provided a kit for identifying a pig, comprising a combination of the primer pairs.

According to still another aspect of the present invention, there is provided a method of detecting DNA comprising: extracting DNA from a sample; Amplifying the extracted DNA by a polymerase chain reaction with the primer pair of claim 1; And analyzing the genotype of the product of the PCR reaction can be provided.

In one embodiment, the polymerase chain reaction is carried out at 95 ° C. for 15 minutes, followed by reaction at 94 ° C. for 40 seconds, 59 to 60 ° C. for 35 to 45 seconds, and 72 ° C. for 35 to 45 seconds. And then reacted at 72 ° C for 10 minutes.

In one embodiment, the cycle of the polymerase chain reaction may be 5 cycles of 40 seconds at 61 DEG C, 5 cycles of 40 seconds at 60 DEG C, and 5 cycles of 40 seconds at 59 DEG C.

In the present invention, in order to detect microsatellite DNA, it is preferable to use a fluorescence probe method rather than a radioactive substance, and to use a fluorescent substance in combination with a primer for gene amplification.

Multiplex PCR was performed by adding 6 μl of template genomic DNA (50 ng / μl), 0.3 μl to 0.4 μl of fluorescence staining primer (10 μl) per set of supersatellite combinations, 2 units / μl of Hot Start Taq DNA polymerase, 2.6 μl, 4 μl of 10 × buffer, and 3 μl of 2.5 mM dNTP were filled with distilled water to give a total reaction volume of 25 μl. The mixture was first denatured at 94 ° C for 40 seconds, bound at 61 ° C for 40 seconds, elongated at 72 ° C for 40 seconds, using a Veriti 96 well Thermal Cycler (Applied Biosystem, CA, USA) Followed by 5 cycles of denaturation at 94 DEG C for 40 seconds, binding at 60 DEG C for 40 seconds, extension at 72 DEG C for 40 seconds, 5 cycles of denaturation at 94 for 40 seconds, binding at 59 DEG C for 40 seconds, extension at 72 DEG C for 40 seconds, Followed by final extension at 72 ° C for 20 minutes. The amplified product after PCR was electrophoresed using ABI-3730 DNA automatic nucleotide sequencer (Applied Biosysytems, USA) to be classified by size and fluorescent substance, and PCR was performed using GeneMapper version 4.0 (Applied Biosystem, USA) The data were collected using Microsoft Excel (USA) program by classifying by product size and type of marker.

In addition, in the present invention, in order to track the origin of pork and to identify individuals, a supersatellite gene used as a genetic marker has 3 to 6 alleles and a heterozygote occurrence rate higher than 60% The size is preferably 81 to 289 bp. If the number of alleles is less than 3, the probability of individual identification becomes too low. If the number of alleles exceeds 6, if the experiment is performed for actual gene analysis, If the size of the gene is 300 bp or more, the detection sensitivity tends to be lowered. If the heterozygote occurrence rate is less than 60%, the allele can not be expressed in various combinations for each individual. Therefore, the number of alleles And it becomes difficult to quantify the size.

Thus, in the present invention, the supersatellite genes SW403, S0226, SW210, SW2410, S0107, S0227, SWR414, S0068, SW1920, SWR2516, S0101, S0018, SW1041, SW1377, SW1557 , SW1989, SW2401, SW2456, SW2515, SW316, SWR1526, SWR1849, and SWR1941 are preferably used. Primer combinations capable of amplifying the 23 genes are listed in SEQ ID NOS: 1 to 46 of the present application.

Hereinafter, the method of tracking the origin of pork and the method for discriminating the pork by gene identification according to the present invention will be described in more detail with reference to examples.

However, these embodiments are only for describing the present invention specifically, and the scope of the present invention is not limited to these embodiments.

[Example]

1. Disclosure Material

The animals and DNA samples used in the present invention are five pigs, consisting of Korean native pigs, Berkshire, Landrace, Yorkshire and Duroc, Genomic DNA was isolated from the blood using a genomic DNA extraction kit (Promega, USA), using 96 clones, 19 clones each, excluding the two clones.

2. Target gene markers and gene amplification primer  making

The present invention relates to a method for gene detection by a multiplex PCR method for identification of a gene in a porcine. The selected microsatellite markers include a microsatellite marker, a Mapviewer database of National Center for Biotechnology Information (NCBI) And allele frequency of the allele genotype, annealing temp. Of the primer, product size of the amplified product, fluorescent substance (Dye), and so on were combined to enable multiplex PCR.

The target gene markers used in the present invention were supersatellite DNA (micro setter), and the loci for analysis of these gene markers are shown in Tables 1 and 2 below.

The names of the genes shown in Tables 1 and 2 are known in the related technical field, for example, on the website (www.NCBI.nlm.nih.gov).

Tables 1 and 2 show the primer combinations for all 23 satellites markers.

chromosome Marker primer
F: Forward (5` 3`)
R: reverse direction (3`5`) Amplification product
size Neon
matter order
number 16 SW403 F GTGTATGTTCATGCATGGGTG 100 ~ 114 Fam One R GTCTCTGCTTTGCTTGCATG 2 2 S0226 F GGTTAAACTTTTNCCCCAATACA 175 - 206 Fam 3 R GCACTTTTAACTTTCATGATGCTCC 4 14 SW210 F TCATCACCATCATACCAAGATG 215 to 250 Fam 5 R AATTCTGCCAAGAAGAGAGCC 6 8 SW2410 F ATTTGCCCCCAAGGTATTTC 102 to 124 VIC 7 R CAGGGTGTGGAGGGTAGAAG 8 4 S0107 F CAAGGATGCCTGTAACTGGTGCAG 165-190 VIC 9 R TCCTTAAGGCCTCGTAGGATCTGT 10 4 S0227 F GATCCATTTATAATTTTAGCACAAAGT 225 to 256 VIC 11 R GCATGGTGTGATGCTATGTCAAGC 12 18 SWR414 F GATTTGACCCCATGCCTG 138 ~ 158 NED 13 R AAGGCAAACCCCTTGAGTTC 14 13 S0068 F AGTGGTCTCTCTCCCTCTTGCT 211 ~ 260 NED 15 R CCTTCAACCTTTGAGCAAGAAC 16 17 SW1920 F GATCCGTATCTATAGCCACCTG 90 to 135 PET 17 R ATGAAAGCTACCAACCCTTCC 18 2 SWR2516 F GTGCATTATCGGGAGGTATG 154-178 PET 19 R ACCCTGTATGATACTGTAACTCTGG 20 7 S0101 F GAATGCAAAGAGTTCAGTGTAGG 197-216 PET 21 R GTCTCCCTCACACTTACCGCAG 22

chromosome Marker primer
F: Forward (5` 3`)
R: reverse direction (3`5`) Amplification product
size Neon
matter order
number 5 S0018 F GCACAGTTGATGCTTCATGC 248-277 NED 23 R GATCAAAAGTCCCCAATTCC 24 10 SW1041 F ATCAGAAAATGGTCAACAGTTCA 93 ~ 103 PET 25 R GGAGAATTCCCAAAGTTAATAGG 26 11 SW1377 F TTCAAGGTTGGAAAGACAGTCC 205 ~ 228 NED 27 R ATGAGGAGTTTGAACTATTGGG 28 14 SW1557 F TTCAAGGTTGGAAAGACAGTCC 84-100 NED 29 R ATGAGGAGTTTGAACTATTGGG 30 15 SW1989 F TGCTCTAATCTACCCGGGTC 228-243 Fam 31 R CCACCCCACTCCCTTCTG 32 9 SW2401 F TGAACAAGTCCAACCAAGAGC 148-170 Fam 33 R CCCAACTAACGGGCTTGTG 34 X SW2456 F GAGCAACCTTGAGCTGGAAC 189-211 PET 35 R AATGTGATTGATGCTGTGAAGC 36 14 SW2515 F CCATCTCATCCAGAAACATCC 90 ~ 108 Fam 37 R AGGATGCTGAGGTGTTAGGC 38 6 SW316 F TTCTCCAGCCATCATGAGTG 133 to 159 PET 39 R AATGACCATTCCTGAGGCTG 40 5 SWR1526 F CGGTGGCTACAGATAACAATAC 114-146 VIC 41 R ATCCGATTCAACCCCTAGC 42 10 SWR1849 F CCTGTTCTGCCTCTAGCCTG 115-160 NED 43 R CTGAGAAGCCTGTGCATCAG 44 13 SWR1941 F AGAAAGCAATTTGATTTGCATAATC 202 ~ 222 VIC 45 R ACAAGGACCTACTGTATAGCACAGG 46

At this time, the optimal annealing temperature (Tm) of the multiplex PCR for the primer combination is 60 ° C in Table 1 and 61 ° C in Table 2, all within 60 ° C to 61 ° C. By designing the base sequence of the primer so that the deviation of the optimum annealing temperature between each primer is minimized, not only the easiness of the chain polymerization reaction but also the accuracy of the reaction result is maximized.

3. Multiplex PCR

PCR amplification was performed mainly by multiplex PCR in consideration of the color of fluorescently dyed supersatellite DNA and distribution of its allele size. PCR was performed for 18 genotypes. At this time, the fluorescent substance was attached to the gene amplification primer in order to recognize the gene tag of the supersagg body.

The multiplex PCR was performed in the presence of about 50 ng of template DNA, 20 ng of each primer, 1.25 mM each dNTP, 0.5 U of Taq DNA polymerase (Promega) and 1 μl of 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, gelatin, 0.25% nonidet P40 and 20 mM MgCl ₂ ) to make the total amount of each reaction solution to 10 μl. Then, the mixture was reacted at 95 ° C. for 15 minutes with a Gene Amp 9700 (GeneAmp 9700, Applied Biosystems) For 40 seconds, 60 ° C for 40 seconds, and 72 ° C for 40 seconds, and the reaction was repeated 25 times, followed by reaction at 72 ° C for 10 minutes. The amplified product after PCR was electrophoresed using ABI-3730 DNA automatic nucleotide sequencer (Applied Biosysytems, USA) to be classified by size and fluorescent substance, and PCR was performed using GeneMapper version 4.0 (Applied Biosystem, USA) The data were collected using Microsoft Excel (USA) program by classifying by product size and type of marker.

Alleles by microsatellite markers determined by Genotyper Software were classified by analysis group and individual using Cervus 3.0 program (version 3.0.3, University of Edinburgh), and then observed for all groups Observed heterozygosity, allele frequency, number of alleles at each locus, and number of alleles at each breed were calculated and are shown in Table 3 below. Table 4 also shows the diversity of alleles of each pig breed by calculating expectation, observed heterozygosity, and polymorphic information content (PIC) values of the specimens.

Table 3 shows the size distribution of alleles and the number of alleles in each gene marker, which can be used as data for individual analysis.

Super satellites
Marker Allele
Size distribution Number of alleles
(all
kind) Number of alleles
(K) Number of alleles
(B) Number of alleles
(L) Number of alleles
(Y) Number of alleles
(D) SW403 100 ~ 114 4 2 3 3 4 2 S0226 175 - 206 5 4 5 5 3 2 SW210 215 to 250 8 3 6 5 3 3 SW2410 102 to 124 6 One One 4 3 3 S0107 165-190 9 3 4 5 5 3 S0227 225 to 256 4 2 2 2 One 3 SWR414 138 ~ 158 4 One 3 2 2 3 S0068 211 ~ 260 9 3 6 5 3 6 SW1920 90 to 135 10 3 6 5 3 6 SWR2516 154-178 7 2 6 4 4 2 S0101 197-216 7 2 4 4 4 3 S0018 248-277 6 2 3 4 3 4 SW1041 93 ~ 103 4 3 2 2 4 2 SW1377 205 ~ 228 4 3 3 2 2 2 SW1557 84-100 6 2 3 6 5 4 SW1989 228-243 8 3 5 4 3 4 SW2401 148-170 7 3 5 5 5 4 SW2456 189-211 5 3 4 5 3 5 SW2515 90 ~ 108 9 2 7 6 5 2 SW316 133 to 159 9 3 5 4 4 3 SWR1526 114-146 5 One 4 3 4 5 SWR1849 115-160 8 3 5 3 4 4 SWR1941 202 ~ 222 9 2 4 5 6 4

K: Korean native, B: Berkshire, L: Landrace, Y: Yorkshire, D: Durok

Table 4 shows the expectation and observed heterozygosity rates and PIC values for pigs of the five varieties of each marker.

Ex H: Expected Heterozygosity

Ob H: Observed heterozygosity

PIC: Polymorphic information content

In the above table, gene diversity can be explained by the expected heterozygosity and is defined as the probability that two randomly selected alleles from the population can exist in different forms. The reference value of the discriminating power in the above index is the PIC value.

By analyzing the 23 alleles of microsatellite markers by multiplex polymerization reaction, it is possible to identify pigs by their high speed and low cost by analyzing various alleles which may appear in pigs, It can be very useful for tracking.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

                         SEQUENCE LISTING <110> RURAL DEVELOPMENT ADMINISTRATION   <120> Discrimination method for product traceability and identification         of pork using Microsatellite DNA <130> NPF24993 <160> 46 <170> PatentIn version 3.2 <210> 1 <211> 21 <212> DNA <213> pork <400> 1 gtgtatgttc atgcatgggt g 21 <210> 2 <211> 20 <212> DNA <213> pork <400> 2 gtctctgctt tgcttgcatg 20 <210> 3 <211> 23 <212> DNA <213> pork <220> <221> misc_feature &Lt; 222 > (13) <223> n is a, c, g, or t <400> 3 ggttaaactt ttnccccaat aca 23 <210> 4 <211> 25 <212> DNA <213> pork <400> 4 gcacttttaa ctttcatgat gctcc 25 <210> 5 <211> 22 <212> DNA <213> pork <400> 5 tcatcaccat cataccaaga tg 22 <210> 6 <211> 21 <212> DNA <213> pork <400> 6 aattctgcca agaagagagc c 21 <210> 7 <211> 20 <212> DNA <213> pork <400> 7 atttgccccc aaggtatttc 20 <210> 8 <211> 20 <212> DNA <213> pork <400> 8 cagggtgtgg agggtagaag 20 <210> 9 <211> 24 <212> DNA <213> pork <400> 9 caaggatgcc tgtaactggt gcag 24 <210> 10 <211> 24 <212> DNA <213> pork <400> 10 tccttaaggc ctcgtaggat ctgt 24 <210> 11 <211> 27 <212> DNA <213> pork <400> 11 gatccattta taattttagc acaaagt 27 <210> 12 <211> 24 <212> DNA <213> pork <400> 12 gcatggtgtg atgctatgtc aagc 24 <210> 13 <211> 18 <212> DNA <213> pork <400> 13 gatttgaccc catgcctg 18 <210> 14 <211> 20 <212> DNA <213> pork <400> 14 aaggcaaacc ccttgagttc 20 <210> 15 <211> 22 <212> DNA <213> pork <400> 15 agtggtctct ctccctcttg ct 22 <210> 16 <211> 22 <212> DNA <213> pork <400> 16 ccttcaacct ttgagcaaga ac 22 <210> 17 <211> 22 <212> DNA <213> pork <400> 17 gatccgtatc tatagccacc tg 22 <210> 18 <211> 21 <212> DNA <213> pork <400> 18 atgaaagcta ccaacccttc c 21 <210> 19 <211> 20 <212> DNA <213> pork <400> 19 gtgcattatc gggaggtatg 20 <210> 20 <211> 25 <212> DNA <213> pork <400> 20 accctgtatg atactgtaac tctgg 25 <210> 21 <211> 23 <212> DNA <213> pork <400> 21 gaatgcaaag agttcagtgt agg 23 <210> 22 <211> 22 <212> DNA <213> pork <400> 22 gtctccctca cacttaccgc ag 22 <210> 23 <211> 20 <212> DNA <213> pork <400> 23 gcacagttga tgcttcatgc 20 <210> 24 <211> 20 <212> DNA <213> pork <400> 24 gatcaaaagt ccccaattcc 20 <210> 25 <211> 23 <212> DNA <213> pork <400> 25 atcagaaaat ggtcaacagt tca 23 <210> 26 <211> 23 <212> DNA <213> pork <400> 26 ggagaattcc caaagttaat agg 23 <210> 27 <211> 22 <212> DNA <213> pork <400> 27 ttcaaggttg gaaagacagt cc 22 <210> 28 <211> 22 <212> DNA <213> pork <400> 28 atgaggagtt tgaactattg gg 22 <210> 29 <211> 22 <212> DNA <213> pork <400> 29 ttcaaggttg gaaagacagt cc 22 <210> 30 <211> 22 <212> DNA <213> pork <400> 30 atgaggagtt tgaactattg gg 22 <210> 31 <211> 20 <212> DNA <213> pork <400> 31 tgctctaatc tacccgggtc 20 <210> 32 <211> 18 <212> DNA <213> pork <400> 32 ccaccccact cccttctg 18 <210> 33 <211> 21 <212> DNA <213> pork <400> 33 tgaacaagtc caaccaagag c 21 <210> 34 <211> 19 <212> DNA <213> pork <400> 34 cccaactaac gggcttgtg 19 <210> 35 <211> 20 <212> DNA <213> pork <400> 35 gagcaacctt gagctggaac 20 <210> 36 <211> 22 <212> DNA <213> pork <400> 36 aatgtgattg atgctgtgaa gc 22 <210> 37 <211> 21 <212> DNA <213> pork <400> 37 ccatctcatc cagaaacatc c 21 <210> 38 <211> 20 <212> DNA <213> pork <400> 38 aggatgctga ggtgttaggc 20 <210> 39 <211> 20 <212> DNA <213> pork <400> 39 ttctccagcc atcatgagtg 20 <210> 40 <211> 20 <212> DNA <213> pork <400> 40 aatgaccatt cctgaggctg 20 <210> 41 <211> 22 <212> DNA <213> pork <400> 41 cggtggctac agataacaat ac 22 <210> 42 <211> 19 <212> DNA <213> pork <400> 42 atccgattca acccctagc 19 <210> 43 <211> 20 <212> DNA <213> pork <400> 43 cctgttctgc ctctagcctg 20 <210> 44 <211> 20 <212> DNA <213> pork <400> 44 ctgagaagcc tgtgcatcag 20 <210> 45 <211> 25 <212> DNA <213> pork <400> 45 agaaagcaat ttgatttgca taatc 25 <210> 46 <211> 25 <212> DNA <213> pork <400> 46 acaaggacct actgtatagc acagg 25

Claims (5)

SEQ ID NOS: 1 and 2, SEQ ID NOS: 3 and 4, SEQ ID NOS: 5 and 6, SEQ ID NOS: 7 and 8, SEQ ID NOS: 9 and 10, SEQ ID NOS: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 28, 29 and 30, SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, And 46 primer pairs,
Wherein the number of alleles is 3 to 6, the heterozygote occurrence rate is higher than 60%, and the size of the genome is 81 to 289 bp. A kit for identifying a conventional pig and a paternity comprising the composition of claim 1. Extracting DNA from the sample;
Subjecting the extracted DNA to a polymerase chain reaction with the composition of claim 1 to amplify the porcine superbody DNA; And
Analyzing the genotype of the polymerase chain reaction product
/ RTI &gt;
Wherein the annealing temperature in the polymerase chain reaction is 60 to 61 ° C. The method of claim 3,
The PCR was carried out at 95 ° C. for 15 minutes, followed by reaction at 94 ° C. for 40 seconds, 60 to 61 ° C. for 35 to 45 seconds, and 72 ° C. for 40 seconds. For 10 minutes. &Lt; / RTI &gt; delete