KR20150056004A - Discrimination method for product traceability and identification of chicken using Microsatellite DNA - Google Patents
Discrimination method for product traceability and identification of chicken using Microsatellite DNA Download PDFInfo
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- KR20150056004A KR20150056004A KR1020130138664A KR20130138664A KR20150056004A KR 20150056004 A KR20150056004 A KR 20150056004A KR 1020130138664 A KR1020130138664 A KR 1020130138664A KR 20130138664 A KR20130138664 A KR 20130138664A KR 20150056004 A KR20150056004 A KR 20150056004A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2531/00—Reactions of nucleic acids characterised by
- C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
- C12Q2531/113—PCR
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
Abstract
The present invention relates to the development of supersatellite markers for genetic identification of chickens, and more particularly, to rapid and economical methods of multiplex chickenpox multiplexing using a microsatellite marker (Multiplex PCR) To identify the individual and to identify the parent. The multi-polymer chain reaction method of the 12 microsatellite markers according to the present invention can be used very usefully for the chicken hysteretic tracer because it is possible to carry out high-speed and low-cost analysis by using the gene for individual identification and paternity identification of the chicken have.
Description
The present invention relates to the development of supersatellite markers for genetic identification of chickens, and more particularly, to rapid and economical methods of multiplex chickenpox multiplexing using a microsatellite marker (Multiplex PCR) To identify the individual and to identify the parent.
In general, tracking the origin of livestock products (chicken) is conducted in order to prevent the spread of malignant diseases such as SARS and poultry typhus to people and animals and animals in other regions in advance. For this purpose, in Europe, only livestock 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, And the origin of livestock products is tracked at the point of sale. In recent years, in order to block malignant infectious agents mediated by chickens, the development of a diagnostic system that can control the origin of chickens and control the logistic flow thereof is being promoted, and the introduction of BT technology .
In addition to the fact that the application of this technology for tracking and tracking origin is being investigated for prevention, there is a very high price difference between the slaughtered livestock and the old breed in Japan and Korea. Therefore, There is a tendency that information of the user is blocked. Even if it is not intentional, it is not linked to perfect origin information due to errors in the logistics system of the slaughtered products of traditional chicken. In addition, it is very important to utilize a system in which information on the birth area of traditional chickens can be delivered in the form of constant logistics information for consumers who choose traditional chicken meat. 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 genomes of chickens vary according to the breeds of chickens, in order to identify individual chickens, markers based on specific genetic patterns of chickens are selected, It is very important to set up the technique.
On the other hand, in Korea, genetic diversity studies on the conservation aspects of chicken genes and breeds, and gene detection techniques using microsatellite DNA (genomic DNA) developed for paternity identification are being used.
However, when the above-mentioned genetic markers are partially used for gene monitoring of chickens, there is a disadvantage in that usefulness and accuracy are inferior.
In addition, it is necessary to develop a sufficient number of genetic markers so as to identify varieties of various individuals while enhancing the effectiveness of chicken identification and origin verification by selecting more useful gene markers. Also, because of the high cost of this analysis, it can be used to monitor the distribution process from time to time, rather than being used in the overall process of distribution, thereby preventing mistakes or intentional product disguises in the distribution process.
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, And to develop an analytical method using the genetic markers of various chickens that can be used to quickly and accurately verify identification and paternity identification.
Accordingly, it is an object of the present invention to provide a method for identifying an individual according to the characteristics of a breed, and selecting a genetic marker from a super-satellite gene of a chicken,
According to an aspect of the present invention, there is provided a method for amplifying supernatant DNA to identify an individual of a chicken, comprising the steps of: 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 .
According to another aspect of the present invention, a chicken identification kit comprising a combination of the above primer pairs may be provided.
According to still another aspect of the present invention, there is provided a method of detecting DNA comprising: extracting DNA from a sample; Amplifying a chicken supernatant DNA by performing a polymerase chain reaction on the extracted DNA with the pair of primers; 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 ° C to 60 ° C for 35 to 45 seconds, and 72 ° C for 40 seconds. The reaction can be repeated at 72 캜 for 10 minutes.
According to one embodiment, the supernatant DNA of the chicken has 3 to 6 alleles, the heterozygote occurrence rate is higher than 60%, and the gene size can be 81 to 289 bp.
The multi-polymer chain reaction method of the 12 microsatellite markers according to the present invention can be used very usefully for the chicken hysteretic tracer because it is possible to carry out high-speed and low-cost analysis by using the gene for individual identification and paternity identification of the chicken have.
In order to accomplish the above object, the present invention provides a method of amplifying a supersatellite gene in a specimen using a gene amplification primer specific to a chicken supersatellite locus, fractionating the amplified gene product by size, And measuring the size of the amplified gene product and comparing the size of the amplified gene product with that of 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 a conventional chicken.
In order to use the supersatellite gene as a genetic marker, in the present invention, genomic DNA was isolated from blood using a 96 genomic DNA extraction kit (Promega, USA).
The present invention relates to a method of gene detection by multiplex PCR for identification of a genome of a chicken, wherein the selected microsatellite markers include a microsatellite marker, a mapviewer database of National Center for Biotechnology Information (NCBI) The frequency of allele frequencies of allele genotypes, annealing temp. Of primers, and size of amplified products were selected based on the loci of super - (Dye), and finally divided into two sets of 23 pairs so that multiplex PCR can be performed.
In this case, the genetic diagnostic primer can prepare two primers per genetic label to analyze the allele gene, perform PCR amplification reaction with the chicken supernatant DNA, and detect each gene marker specifically expressed in the chicken 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 occurs at a high frequency in a chicken population from the above selection method.
According to an aspect of the present invention, there is provided a method for amplifying supernatant DNA to identify an individual of a chicken, comprising the steps of: 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 .
According to another aspect of the present invention, a chicken identification kit comprising a combination of the above primer pairs may be provided.
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.
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 40 seconds, and 72 ° C. for 40 seconds, And allowed to react at 72 ° C for 10 minutes.
In one embodiment, the polymerase chain reaction was performed by first denaturing at 95 ° C for 15 min, denaturation at 94 ° C for 40 sec, binding at 61 ° C for 40 sec, elongation at 72 ° C for 40 sec, After 25 cycles of denaturation for 40 seconds, binding at 60 DEG C for 40 seconds, elongation at 72 DEG C for 40 seconds, 5 cycles, denaturation at 94 DEG C for 40 seconds, binding at 59 DEG C for 40 seconds, elongation at 72 DEG C for 40 seconds, Lt; RTI ID = 0.0 > 72 C < / RTI > for 20 minutes.
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 using 6ul of template genomic DNA (50ng / ul), 0.3μl - 0.4μl of Hot Start Taq DNA Polymerase (2Unit / μl) per set of fluorescent dye primer (10pmole) 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 DEG C for 40 seconds, binding at 59 DEG C for 40 seconds, Cycle, and finally a final extension reaction at 72 캜 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, the supersatellite gene used as a genetic marker for the purpose of tracking the origin of the chicken and identifying the individual is 3 to 6 alleles and the heterozygote occurrence rate is 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.
Therefore, in the present invention, it is preferable to use MCW0123, MCW0078, LEI094, ADL0268, MCW0069, MCW0111, MCW0034, MCW0295, MCW0216, MCW0248, MCW0330, LEI0166 which are suited to the above conditions and have a high appearance frequency in chicken .
Hereinafter, the method of tracking the origin of the meat and the method for discriminating the breed by the gene detection of 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
Genomic DNA was isolated from the blood using a genomic DNA extraction kit (Promega, USA) using 96 clones from the wild animals and DNA samples used in the present invention.
2. Target gene markers and gene amplification primer making
The present invention relates to a method of gene detection by multiplex PCR for identification of a genome of a chicken, wherein the selected microsatellite markers include a microsatellite marker, a mapviewer database of National Center for Biotechnology Information (NCBI) The frequency of allele frequencies of allele genotypes, annealing temp. Of primers, and size of amplified products were selected based on the loci of super - (Dye), and so on were combined to enable multiplex PCR.
The target gene markers used in the present invention are supersatellite DNA (micro setter), and the loci for analyzing these gene markers are shown in Table 1 below.
The genetic markers listed in Table 1 are conventionally known in the technical field, for example, on the website (www.NCBI.nlm.nih.gov).
Table 1 shows primer combinations for the 12 satellites markers.
F: Forward (5` 3`)
R: reverse direction (3`5`)
size
matter
number
The optimal annealing temperature (Tm) for multiplex PCR for this primer combination is all within 59-60 ° 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 carried out 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, 0.01 % Gelatin, 0.25% nonidet P40 and 20 mM MgCl 2 ) to make the total amount of each reaction solution to 10 μl. The mixture was reacted at 95 ° C. for 15 minutes with a Gene Amp 9700 (GeneAmp 9700, Applied Biosystems) 40 seconds at 60 ° C, 40 seconds at 60 ° C, and 40 seconds at 72 ° C, and the reaction was repeated at 25 ° C for 10 minutes at 72 ° C. 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 2 below. Table 3 shows the diversity of the allelic genotypes of the strains by calculating the expectation, observed heterozygosity, and polymorphic information content (PIC) values for the 96 samples.
Table 2 shows the size distribution of alleles and the number of alleles for each gene marker, which can be used as data for individual analysis.
Table 3 shows the expectation and observed heterozygosity rates and PIC values for each chicken 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 criteria for distinguishing power in the above index are PIC values in the order of MCW0330>ADL0268>MCW0034>LEI0166>MCW0248>MCW0123>MCW0069>MCW0111>MCW0216>MCW0078>MCW0295> LEI0094.
Since the above 12 microsatellite markers can be amplified by multiplex polymerization to analyze various alleles which may appear in a chicken, it is possible to identify the individual and identify the individual with high speed and low cost, 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 chicken using Microsatellite DNA <130> NPF24992 <160> 24 <170> PatentIn version 3.2 <210> 1 <211> 21 <212> DNA <213> chicken <400> 1 ccactagaaa agaacatcct c 21 <210> 2 <211> 21 <212> DNA <213> chicken <400> 2 ggctgatgta agaagggatg a 21 <210> 3 <211> 22 <212> DNA <213> chicken <400> 3 ccacacggag aggagaaggt ct 22 <210> 4 <211> 24 <212> DNA <213> chicken <400> 4 tagcatatga gtgtactgag cttc 24 <210> 5 <211> 21 <212> DNA <213> chicken <400> 5 gatctcacca gtatgagctg c 21 <210> 6 <211> 21 <212> DNA <213> chicken <400> 6 tctcacactg taacacagtg c 21 <210> 7 <211> 20 <212> DNA <213> chicken <400> 7 ctccacccct ctcagaacta 20 <210> 8 <211> 20 <212> DNA <213> chicken <400> 8 caacttccca tctacctact 20 <210> 9 <211> 22 <212> DNA <213> chicken <400> 9 gcactcgaga aaacttcctg cg 22 <210> 10 <211> 24 <212> DNA <213> chicken <400> 10 attgcttcag caagcatggg agga 24 <210> 11 <211> 20 <212> DNA <213> chicken <400> 11 gctccatgtg aagtggttta 20 <210> 12 <211> 20 <212> DNA <213> chicken <400> 12 atgtccactt gtcaatgatg 20 <210> 13 <211> 24 <212> DNA <213> chicken <400> 13 tgcacgcact tacatactta gaga 24 <210> 14 <211> 24 <212> DNA <213> chicken <400> 14 tgtccttcca attacattca tggg 24 <210> 15 <211> 21 <212> DNA <213> chicken <400> 15 atcactacag aacaccctct c 21 <210> 16 <211> 21 <212> DNA <213> chicken <400> 16 tatgtatgca cgcagatatc c 21 <210> 17 <211> 20 <212> DNA <213> chicken <400> 17 gggttttaca ggatgggacg 20 <210> 18 <211> 20 <212> DNA <213> chicken <400> 18 agtttcactc ccagggctcg 20 <210> 19 <211> 22 <212> DNA <213> chicken <400> 19 gttgttcaaa agaagatgca tg 22 <210> 20 <211> 21 <212> DNA <213> chicken <400> 20 ttgcattaac tgggcacttt c 21 <210> 21 <211> 21 <212> DNA <213> chicken <400> 21 tggacctcat cagtctgaca g 21 <210> 22 <211> 22 <212> DNA <213> chicken <400> 22 aatgttctca tagagttcct gc 22 <210> 23 <211> 20 <212> DNA <213> chicken <400> 23 ctcctgccct tagctacgca 20 <210> 24 <211> 20 <212> DNA <213> chicken <400> 24 tatcccctgg ctgggagttt 20
Claims (5)
Amplifying a chicken supernatant DNA by performing a polymerase chain reaction on the extracted DNA with the primer pair of claim 1; And
Analyzing the genotype of the polymerase chain reaction product
≪ / RTI >
The PCR was 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. For 10 minutes. ≪ Desc / Clms Page number 20 >
Wherein the chicken supernatant DNA has 3 to 6 alleles, a heterozygote occurrence ratio is higher than 60%, and a gene size is 81 to 289 bp.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170027078A (en) * | 2015-09-01 | 2017-03-09 | 고려대학교 산학협력단 | Composition for discrimination of Yeonsan Ogye |
| KR20180050470A (en) * | 2016-11-04 | 2018-05-15 | 한경대학교 산학협력단 | Microsatellite Marker For Identification of Korean Native Chicken And Method For Identification Using The Same |
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2013
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170027078A (en) * | 2015-09-01 | 2017-03-09 | 고려대학교 산학협력단 | Composition for discrimination of Yeonsan Ogye |
| KR20180050470A (en) * | 2016-11-04 | 2018-05-15 | 한경대학교 산학협력단 | Microsatellite Marker For Identification of Korean Native Chicken And Method For Identification Using The Same |
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