KR20190041798A - Composition for discrimination of sex and breed for cattle, and discriminating method using the same - Google Patents

Abstract

The present invention relates to a method and composition capable of determining sex and breed, distinguishing individuals, and determining parentage of cattle more quickly, accurately and economically than conventional methods, through multiplex PCR using a microsatellite marker and a sex chromosome marker for determining the sex and breed of cattle.

Description

TECHNICAL FIELD The present invention relates to a composition for discriminating cattle breeds and breeds, and a discriminating method using the same.

TECHNICAL FIELD The present invention relates to a composition for the identification of a bovine sexuality and a variety, and a discriminating method using the same. More particularly, the present invention relates to a method and apparatus for rapid, accurate, and economical identification of bovine germs through multiplex PCR using sex chromosome marker and microsatellite markers for bovine sex and breed identification, The present invention relates to compositions and methods capable of breed, individual identification, paternity, and the like.

Consumers' demands for more hygienic and safer livestock products are rapidly increasing as consumers are becoming more anxious about beef due to recent outbreaks of mad cow disease and foot - and - mouth disease.

However, in some restaurants, there is an increasing number of instances in which imported cattle or domestic beef cattle are sold as cattle, resulting in unreasonable damages to sincere producers, It is in disturbance. In addition, with the import of foreign beef, the domestic Hanwoo industry structure is absolutely lacking in competitiveness to survive competition with cheap imports.

Therefore, in order to improve the transparency of livestock distribution by recording and managing transactions on livestock distribution, and to contribute to the healthy development of livestock industry and related industries by preventing false sales of false markings of origin .

Genetic markers using biotechnology continue to be developed and used to identify varieties and sex for a variety of plants and animals. Especially, in the livestock industry, the control of male and female sex ratio is one of the most important factors that determines the scale of the marine and the class of the marine.

Therefore, it is necessary to improve the technical ability to complement the DNA identity test related to the livestock traceability agent, and it is necessary to identify the breed more quickly and accurately, and to analyze the paternity feeling and sex discrimination.

As a background technique of the present invention, there is Korean Patent Registration No. 10-1360238 (Apr. 21, 2014).

A number of patent documents are referenced and cited throughout the present invention. The disclosures of the cited patent documents are hereby incorporated by reference into the present invention in its entirety to better illustrate the state of the art to which the present invention pertains and the content of the present invention.

It is an object of the present invention to provide a sex chromosome marker and a microsatellite marker that enable rapid, accurate and economical identification of sex, breed, individual identification, and paternity.

It is another object of the present invention to provide a primer set capable of amplifying the microsatellite markers and sex chromosome marker, and a composition comprising the primer set.

It is still another object of the present invention to provide a kit comprising the primer set.

It is still another object of the present invention to provide a method for determining the quality and kind of cattle using the composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, a first primer set of SEQ ID NOS: 1 and 2; And a second primer set of SEQ ID NOS: 3 and 4; And a primer set of at least one of the primer sets.

According to one embodiment of the present invention, there is provided a composition for determining sex of a bovine including a first primer set and a second primer set.

According to one embodiment of the present invention, a third primer set of SEQ ID NOS: 5 and 6; A fourth primer set of SEQ ID NOS: 7 and 8; A fifth primer set of SEQ ID NOS: 9 and 10; A sixth primer set of SEQ ID NOS: 11 and 12; A seventh primer set of SEQ ID NOS: 13 and 14; An eighth primer set of SEQ ID NOS: 15 and 16; A ninth primer set of SEQ ID NOS: 17 and 18; A tenth primer set of SEQ ID NOS: 19 and 20; An eleventh primer set of SEQ ID NOS: 21 and 22; A twelfth primer set of SEQ ID NOS: 23 and 24; A thirteenth primer set of SEQ ID NOS: 25 and 26; A fourteenth primer set of SEQ ID NOS: 27 and 28; And a fifteenth primer set of SEQ ID NOs: 29 and 30, to thereby further determine the kind of the bovine animal.

According to another aspect of the present invention, there is provided a kit for the determination of cattle sex, which comprises a composition for determining the quality of cattle.

According to another aspect of the present invention, there is provided a method for detecting nucleic acid, comprising the steps of: Multiplex PCR amplification using the above-described primer set using the extracted nucleic acid as a template; And detecting the allele by electrophoresis of the amplified product in the amplifying step and analyzing the size to discriminate the sex and the breed.

According to an embodiment of the present invention, it can be determined that the amplified product is hydrogen when it is 216bp and 156bp.

According to still another aspect of the present invention, there is provided a primer set comprising: a first primer set of SEQ ID NOS: 1 and 2; A second primer set of SEQ ID NOS: 3 and 4; A third primer set of SEQ ID NOS: 5 and 6; A fourth primer set of SEQ ID NOS: 7 and 8; A fifth primer set of SEQ ID NOS: 9 and 10; A sixth primer set of SEQ ID NOS: 11 and 12; A seventh primer set of SEQ ID NOS: 13 and 14; An eighth primer set of SEQ ID NOS: 15 and 16; A ninth primer set of SEQ ID NOS: 17 and 18; A tenth primer set of SEQ ID NOS: 19 and 20; An eleventh primer set of SEQ ID NOS: 21 and 22; A twelfth primer set of SEQ ID NOS: 23 and 24; A thirteenth primer set of SEQ ID NOS: 25 and 26; A fourteenth primer set of SEQ ID NOS: 27 and 28; And a fifteenth primer set of SEQ ID NOs: 29 and 30 are provided.

According to another aspect of the present invention, there is provided a method for detecting nucleic acid, comprising the steps of: Performing multiplex PCR amplification using the extracted nucleic acid as a template and using the primer set of SEQ ID NOS: 1 to 30; And a step of detecting alleles of the amplified products through the electrophoresis apparatus and analyzing the sizes thereof to discriminate the sex and the breed of the cattle.

According to one embodiment of the present invention, the cow is provided with a method for discriminating the sex and the breed of cattle, which comprises Yeongam Hanwoo, Jangheung Hanwoo, Brown Swiss, Limousine, Angus, Simmental, Hairford, Sharule or Holstein.

According to one embodiment of the present invention, it is possible to simultaneously and rapidly discriminate cow and male sex by PCR amplification alone. Therefore, the present invention can be applied to the production of animals adapted for male and female through somatic cell replication and embryo transfer.

According to one embodiment of the present invention, sex discrimination, breed identification, individual identification, and paternity detection are possible in a faster, more accurate and economical manner.

According to one embodiment of the present invention, it can be very usefully used for technical improvement of the beef trace agent and supplementation of the traceability system.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context clearly indicates otherwise, the singular form of the term also includes plural forms thereof, and plural forms of the term should be understood as including its singular form.

According to an aspect of the present invention, a first primer set of SEQ ID NOS: 1 and 2; And a second primer set of SEQ ID NOS: 3 and 4; And a primer set of at least one of the primer sets.

The CattleX_1 and / or CattleY_2 gene markers of the present invention can be rapidly and accurately discriminated by PCR amplification. Therefore, the present invention can be applied to the production of animals adapted for male and female through somatic cell replication and embryo transfer.

As used herein, the term " primer " refers to a short sequence that serves as a starting point for template strand replication as a base sequence having a short free 3 'hydroxyl group. The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

According to one embodiment of the present invention, a first primer set and a second primer set may be included. With this configuration, sex discrimination can be performed more accurately.

According to one embodiment of the present invention, a third primer set of SEQ ID NOS: 5 and 6; A fourth primer set of SEQ ID NOS: 7 and 8; A fifth primer set of SEQ ID NOS: 9 and 10; A sixth primer set of SEQ ID NOS: 11 and 12; A seventh primer set of SEQ ID NOS: 13 and 14; An eighth primer set of SEQ ID NOS: 15 and 16; A ninth primer set of SEQ ID NOS: 17 and 18; A tenth primer set of SEQ ID NOS: 19 and 20; An eleventh primer set of SEQ ID NOS: 21 and 22; A twelfth primer set of SEQ ID NOS: 23 and 24; A thirteenth primer set of SEQ ID NOS: 25 and 26; A fourteenth primer set of SEQ ID NOS: 27 and 28; And a fifteenth primer set of SEQ ID NOs: 29 and 30, to thereby further determine the kind of the bovine animal.

According to the above-described configuration, not only the determination of the quality of cattle but also the breed of cattle, individual identification and paternity can be performed at the same time.

According to another aspect of the present invention, there is provided a kit for determining the quality of a beef comprising the composition for determining the quality of a beef cattle of the present invention.

When the kit of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally comprise a reagent, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus (Tth) Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu), DNA polymerase joins and dNTPs, and where the kit of the invention is applied to immunoassay, May optionally include a secondary antibody and a labeling substrate. Further, the kit according to the present invention may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

According to another aspect of the present invention, there is provided a method for detecting nucleic acid, comprising the steps of: Amplifying the nucleic acid using the extracted nucleic acid as a template and using the primer set; And a step of detecting alleles of the amplified product through the electrophoresis apparatus and analyzing the size to discriminate gender.

According to an embodiment of the present invention, it can be determined that the amplified product is hydrogen when it is 216bp and 156bp. Further, when the amplified product is 216bp, it can be discriminated as a cow.

According to still another aspect of the present invention, there is provided a primer set comprising: a first primer set of SEQ ID NOS: 1 and 2; A second primer set of SEQ ID NOS: 3 and 4; A third primer set of SEQ ID NOS: 5 and 6; A fourth primer set of SEQ ID NOS: 7 and 8; A fifth primer set of SEQ ID NOS: 9 and 10; A sixth primer set of SEQ ID NOS: 11 and 12; A seventh primer set of SEQ ID NOS: 13 and 14; An eighth primer set of SEQ ID NOS: 15 and 16; A ninth primer set of SEQ ID NOS: 17 and 18; A tenth primer set of SEQ ID NOS: 19 and 20; An eleventh primer set of SEQ ID NOS: 21 and 22; A twelfth primer set of SEQ ID NOS: 23 and 24; A thirteenth primer set of SEQ ID NOS: 25 and 26; A fourteenth primer set of SEQ ID NOS: 27 and 28; And a fifteenth primer set of SEQ ID NOs: 29 and 30 are provided.

According to another aspect of the present invention, there is provided a method for detecting nucleic acid, comprising the steps of: Performing multiplex PCR amplification using the extracted nucleic acid as a template and using the primer set of SEQ ID NOS: 1 to 30; And a step of detecting alleles of the amplified products through the electrophoresis apparatus and analyzing the sizes thereof to discriminate the sex and the breed of the cattle.

According to one embodiment of the present invention, the cow is provided with a method for discriminating the sex and the breed of cattle, which comprises Yeongam Hanwoo, Jangheung Hanwoo, Brown Swiss, Limousine, Angus, Simmental, Hairford, Sharule or Holstein.

In the present invention, the step of separating the nucleic acid from the target small sample may be performed by a method known in the art. For example, DNA can be directly purified from tissues or cells or amplified by specific amplification of specific regions using amplification methods such as PCR and separation thereof. In the present invention, nucleic acid means not only DNA but also cDNA and RNA molecules synthesized from mRNA. The step of extracting DNA from the target sample can be performed by, for example, PCR amplification, ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 USA 86, 1173 (1989)) and self-sustained sequence cloning (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)), as well as transcription amplification (Kwoh et al., Proc. Natl. Acad. And nucleic acid-based sequence amplification (NASBA).

In the present invention, PCR can be performed using a PCR reaction mixture containing various components known in the art required for PCR reaction. In addition to the DNA extracted from the sample to be analyzed and the primer set provided in the present invention, the PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH ₂ O). The PCR buffer comprises Tris -HCl (Tris-HCl), MgCl 2, KCl and the like. At this time, the MgCl ₂ concentration greatly affects the specificity and yield of the amplification, preferably 1.5-2.5 mM. In general, when Mg ²⁺ is excessive, nonspecific PCR amplification products are increased, and when Mg ²⁺ is insufficient, the yield of PCR products is decreased. The PCR buffer solution may further contain an appropriate amount of Triton X-100 (Triton X-100).

In the present invention, the PCR conditions consisted of denaturation at 94 DEG C for 60 seconds, binding at 60 DEG C for 60 seconds, extension at 72 DEG C for 60 seconds, denaturation at 94 DEG C for 60 seconds, After incubation for 60 seconds at 72 ° C for 60 seconds, 5 cycles of denaturation at 94 ° C for 60 seconds, binding at 58 ° C for 60 seconds, extension at 72 ° C for 60 seconds, and finally 25 cycles of final elongation at 65 ° C for 30 minutes Reaction can be carried out, but can be carried out according to ordinary methods which can be understood by the ordinary skill in the art, so that it is not particularly limited.

In the present invention, size-specific analysis of the DNA of the PCR amplification product can be performed according to methods well known in the art. Preferably by agarose gel or polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3730 genetic analyzer-electropherogram). At this time, in order to use the fluorescence analyzer, the PCR is performed using a primer set with a fluorescent dye known in the art.

In the meantime, the present invention provides a gene detection method using a multiplex PCR method for gene identification / paternity detection and sex determination of bovine animals. The selected satellites markers drive a Tandem Repeat Finder program based on information on the next generation nucleotide sequence microsatellite genetic loci were selected and sex discrimination was performed using the differences in the length of the ZFP gene sequence on the sex chromosome. The detailed selection criteria were Allele Frequency, Annealing Temp., Annealing Temp. (Product size), fluorescence (Dye), etc. Finally, a total of 15 gene markers were combined to enable multiplex PCR.

&Quot; Microsatellites " as used herein refers to short repeating sequences in the form of mono-, di-, tri-, and tetra-nucleotides distributed throughout the eukaryotes gene. Generally, these repeating sequences exist in a tandem repeat form about 10 to 40 times in the chromosome.

As used herein, " microsatellite marker " means a DNA polymorphism detection marker using microsatellite. Since each individual has specificity in the genotype combination of markers, a sufficient number of markers can be used to identify the individual as well as the breed. In order to improve the accuracy and speed of gene detection, many surveillance markers should be investigated. In case of performing gene detection on many supersatellite, There was a problem of consuming manpower. Therefore, in order to solve this problem, a multiplex PCR method in which a primer of several marker factors is inserted into a single PCR reaction and a reaction is performed simultaneously is useful.

The marker composition of the present invention comprises BZSY, BZSX, BHXC29, BPC113, BPC115, BPC19, BPC21, BPC7, BTEC17, BTEC4, BTRC11, BTRC16, BTRC19, BTRC6 and BTRC9, Lt; RTI ID = 0.0 > 30 < / RTI >

According to one embodiment of the present invention, the cow is provided with a method for discriminating the sex and the breed of cattle, which comprises Yeongam Hanwoo, Jangheung Hanwoo, Brown Swiss, Limousine, Angus, Simmental, Hairford, Sharule or Holstein.

Table 1 shows the bovine genetic markers (two sex markers and thirteen satellites markers) and shows the chromosomal location, the primer base sequence, the amplification product size, the fluorescent staining method, and the SEQ ID NO.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example

The disclosure materials used in the present invention are as follows: Yeongam Hanwoo 30, Jangheung Han 30, Brown Swiss 19, Angus 18, Anghem 18, ) And Holstein (10 strains). Genomic DNA was isolated from 185 blood samples of 9 different cultivars using genomic DNA extraction kit (Promega, USA).

Multiplex PCR was performed using 4 μl of template genomic DNA (20 ng / μl), 0.3 μl-0.4 μl per set of fluorescence staining primers (10 pmole) of each supersatellite combination, 2 units / μl of Hot Start Taq DNA polymerase ), 3 占 퐇 of 10X buffer, and 2.4 占 퐇 of 2.5 mM dNTP were filled with distilled water to make the total reaction solution 20 占 퐇. The mixture was denatured at 94 ° C for 60 seconds, bound at 60 ° C for 60 seconds, initiated at 95 ° C for 15 minutes using Thermal Cycler PTC-0240 (MJ Research, Inc., MA, USA) 60 sec., Denaturation at 94 deg. C for 60 sec, binding at 59 deg. C for 60 sec, extension at 72 deg. C for 60 sec, 5 cycles, denaturation at 94 deg. C for 60 sec, binding at 58 deg. C for 60 sec and elongation at 72 deg. C for 60 sec After 25 cycles, the final elongation reaction was performed at 65 ° C for 30 minutes.

The amplified product after PCR was electrophoresed using ABI-3730 DNA automatic nucleotide sequencer (Applied Biosytems, USA) to be classified according to 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.

Finally, the alleles of all microsatellite markers determined by Genotyper Software were grouped by analysis group and individual using microsatellite toolkit software (Park, 2000) heterozygosity, and allele counts were calculated (Table 2). Table 2 shows the mean and standard error of the heterozygosity of each species of cattle and the average number of alleles. Table 3 shows the amplification product size, number of alleles and heterozygosity of each species of supersaturate marker.

In addition, the expectation and observed heterozygosity rates and PIC of the 9 varieties of each markers showed various values (Table 4). The diversity of thirteen satellites was verified through the expectation and observed heterozygosity rate and PIC value of each marker, and it was confirmed that it could be used as individual marker or paternity emotional marker. Table 4 shows the expected and observed heterozygosity rates and PIC values for the 9 varieties of each marker.

The bovine sex-specific marker was amplified to 216 bp in the X chromosome and amplified in 156 bp in the Y chromosome. In the case of hydrogen (♂), two peaks were observed at 216 bp and 156 bp since it was XY. In the case of cow, , 216 bp, and the genotype was expressed with one peak (Table 5). Table 5 shows the amplification product size (bp) of the bovine sex-determining gene marker.

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.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Composition for discrimination of sex and breed for cattle, and          discriminating method using same <130> NPF-31514 <160> 30 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 1 agcagttggg agtcacatgg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 2 ggctcaccat gtcctccttc 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 cagcatcctg taagggttat ttg 23 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 4 ccccatggac tctcttgttg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 5 ctcccatcga gctctttcac 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 6 cctacctggc agaggacaaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 7 attccaccca aaaccagtca 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 8 actcattgtc tgaagtgaaa agca 24 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 9 gcaagaggtc cagatggtgt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 10 agagaacaac aaggcgaagc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 11 atgaggtggg agaaaagcaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 12 ttctcaggag tacgcaagca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 13 tggatccttt ttggctctgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 14 agcactggca ggtactggag 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 15 ccctgacttc cagatcctgt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 16 aacccagact ggttctgctt 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 17 ttcctggttc tccaatttgc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 18 ggctttcaaa aggctcaaca 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 19 gggattacat aagtcggtgg a 21 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 20 tgaggcaatc aaaagtccaa 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 21 cgagagctgc acctaccttc 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 22 cccgagctct ctttcccta 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 23 aagcaaggga aatcattcca 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 24 cacctacctg agcggagaag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 25 ccatgcctat tttgtgctga 20 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 26 actgaggtca agtagggtga tagaa 25 <210> 27 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 27 tggaattttc tttgatagaa ttagcc 26 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 28 tttcccagca gtttttaacg a 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 29 tccacagttt gcttagccat t 21 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 30 ggtgtggctc tgtgttcctt 20

Claims (9)

A first primer set of SEQ ID NOS: 1 and 2; And
A second primer set of SEQ ID NOS: 3 and 4; Wherein the primer set comprises at least one primer set. The composition according to claim 1, comprising a first primer set and a second primer set. The method according to claim 1,
A third primer set of SEQ ID NOS: 5 and 6; A fourth primer set of SEQ ID NOS: 7 and 8; A fifth primer set of SEQ ID NOS: 9 and 10; A sixth primer set of SEQ ID NOS: 11 and 12; A seventh primer set of SEQ ID NOS: 13 and 14; An eighth primer set of SEQ ID NOS: 15 and 16; A ninth primer set of SEQ ID NOS: 17 and 18; A tenth primer set of SEQ ID NOS: 19 and 20; An eleventh primer set of SEQ ID NOS: 21 and 22; A twelfth primer set of SEQ ID NOS: 23 and 24; A thirteenth primer set of SEQ ID NOS: 25 and 26; A fourteenth primer set of SEQ ID NOS: 27 and 28; And a 15th primer set of SEQ ID NOs: 29 and 30, wherein the composition for distinguishing bovine can be further distinguished from a bovine. A kit for the determination of the quality of cattle comprising the composition for determining the quality of cattle as claimed in any one of claims 1 to 3. Extracting the nucleic acid from the target small sample;
Multiplex PCR amplification using the extracted nucleic acid as a template and using the primer set described in any one of claims 1 to 3; And
Detecting the allele by electrophoresis of the amplified product in the amplifying step, and analyzing the size to discriminate the sex and the cultivar. 6. The method of claim 5,
And determining that the amplified product is hydrogen when the amplified product is 216 bp and 156 bp. A first primer set of SEQ ID NOS: 1 and 2; A second primer set of SEQ ID NOS: 3 and 4; A third primer set of SEQ ID NOS: 5 and 6; A fourth primer set of SEQ ID NOS: 7 and 8; A fifth primer set of SEQ ID NOS: 9 and 10; A sixth primer set of SEQ ID NOS: 11 and 12; A seventh primer set of SEQ ID NOS: 13 and 14; An eighth primer set of SEQ ID NOS: 15 and 16; A ninth primer set of SEQ ID NOS: 17 and 18; A tenth primer set of SEQ ID NOS: 19 and 20; An eleventh primer set of SEQ ID NOS: 21 and 22; A twelfth primer set of SEQ ID NOS: 23 and 24; A thirteenth primer set of SEQ ID NOS: 25 and 26; A fourteenth primer set of SEQ ID NOS: 27 and 28; And a fifteenth primer set of SEQ ID NOs: 29 and 30, respectively. Extracting the nucleic acid from the target small sample;
Multiplex PCR amplification using the extracted nucleic acid as a template and using the primer set described in claim 7; And
Detecting the allele of the amplified product in the amplifying step through the electrophoresis apparatus and analyzing the size thereof to discriminate the sex and the breed of the cattle. 9. The method of claim 8,
Wherein the cow is Young Han Hanwoo, Jangheung Hanwoo, Brown Swiss, Limousine, Angus, Simmental, Hairford, Sharrile or Holstein.