KR20070005401A - The primers specific to cervus elaphus, c. nippon, c. canadensis and rangifer tarandus gene and the method to identify cervi parvum cornu species - Google Patents

Abstract

Primers specific to Cervus elaphus, C. nippon, C. canadensis and Rangifer tarandus genes and a method for identifying Cervi parvum Cornu species by using the same primers are provided to improve identification accuracy of Cervi parvum Cornu, and prevent illegal distribution of horns of other animals except Cervi parvum Cornu. The reverse primers specific to D-loop of mitochondria genes of Cervus elaphus, C. nippon, C. canadensis and Rangifer tarandus have the nucleotide sequences of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively. The forward primer specific to D-loop of mitochondria genes of Cervus elaphus, C. nippon, C. canadensis and Rangifer tarandus has the nucleotide sequence of SEQ ID NO:4. The method for identifying Cervi parvum Cornu species comprises the steps of: (1) extracting DNA from a subject; (2) performing multiplex-PCR by using the extracted DNA as a template and primers of Cervus elaphus, C. nippon, C. canadensis and Rangifer tarandus genes; (3) subjecting the PCR products to electrophoresis; and (4) verifying the PCR products.

Description

The primers specific to Cervus elaphus, C. nippon, C. canadensis and Rangifer tarandus gene and the method to The primers specific to Cervus elaphus, C. nippon, C. canadensis and Rangifer tarandus gene and the method to identify Cervi Parvum Cornu species}

1 is a diagram showing the results of PCR with a template of the mitochondrial gene D-loop of red deer, Japanese deer, elk and reindeer,

1, Cervus elaphus (RED); 2, C. nippon (NIP); 3, C. elaphus canadensis (ELK); 4, Rangifer tarandus (REIN); L, 100bp ladder.

FIG. 2 is a diagram showing multiple alignment of D-loop sites amplified for each species and primers specifically designed for the species.

A, forward primer (FOR); B, RED primer; C, ELK primers; D, SIKA primer; E, REIN primer.

Figure 3 is a diagram showing the results of PCR using a mitochondrial D-loop of each species using a species-specific primer,

1, Cervus elaphus ; 2, Cervus nippon ; 3, Cervus elaphus canadensis ; 4, Rangifer tarandus ; 5, mouse; 6, rat; 7, cats; 8, cattle; 9, dog; 10, person; 11, negative control; L, 100bp ladder.

FIG. 4 is a diagram showing PCR amplification sensitivity when PCR is carried out using a mitochondrial D-loop of each species using a species-specific primer.

A. RED B. NIP C. ELK D. REIN.

1, 1 ng; 2, 500 pg; 3, 100 pg; 4, 50 pg; 5, 10 pg;

6, 5 pg; 7, 1 pg; 8, 0.5 pg; L, 100bp ladder

5 is a diagram showing a result of comparing the patterns of amplification products when multiplex-PCR was performed with a species-specific primer on a sample mixed with DNA of each deer species,

1, Cervus elaphus : Cervus nippon = 1: 1;

2, Cervus elaphus : Cervus canadensis . = 1: 1;

3, Cervus elaphus : Rangifer tarnadus = 1: 1;

4, Cervus nippon : Cervus canadensis = 1: 1;

5, Cervus nippon : Rangifer tarnadus = 1: 1;

6, Cervus canadensis : Rangifer tarnadus = 1: 1;

7, Cervus elaphus : Cervus nippon : Cervus canadensis = 1: 1: 1;

8, Cervus elaphus : Cervus nippon : Rangifer tarnadus = 1: 1: 1;

9, Cervus elaphus : Cervus canadensis : Rangifer tarnadus = 1: 1: 1; 10, Cervus nippon : Cervus canadensis : Rangifer tarnadus = 1: 1: 1; 11, Cervus elaphus : Cervus nippon : Cervus canadensis : Rangifer tarnadus = 1: 1: 1: 1; L, 100bp ladder

6 is a diagram showing a result of comparing the patterns of amplification products when multiplex-PCR was performed with a species-specific primer on a sample mixed with DNA of each deer and reindeer.

A. Cervus elaphus (a): Rangifer tarnadus (b) .

B. Cervus nippon (a): Rangifer tarnadus (b).

C. Cervus elaphus canadensis (a) Rangifer tarnadus (b).

1, a: b = 1: 14; 2, a: b = 1: 9; 3, a: b = 3: 7; 4, a: b = 5: 5; 5, a: b = 7: 3;

6, a: b = 9: 1; 7, a: b = 14: 1; L, 100bp ladder

7 is a diagram showing the results of performing multiplex-PCR with species-specific primers on the antler samples distributed in the market.

1, Korea (Elk); 2, New Zealand; 3, Russia; 4, Korea (import 1);

5, Korea (import 2); 6, China; 7, New Zealand; 8, Russia; 9, Canada; 10, China; 11, China, 12, Rangifer tarandus ; 13, negative control (DW); L, 100bp ladder

The present invention relates to a primer specific for a red deer ( Cervus elaphus ), a Japanese deer ( C. nippon ), an elk ( C. canadensis ), and a reindeer ( Rangifer tarandus ) and a method for identifying a antler variety using the primers. More specifically, the present invention relates to individual primers having sequences specific to the genes of Japanese deer, red deer, elk and reindeer, and a method of identifying antler varieties by multiplex-PCR using the primers.

Deer antler (Cervi Parvum Cornu) is a cut of the coccyx of the subspecies ( C. elaphus L.) or elk ( C. elaphus canadensis ) of the Japanese deer ( Cervus nippon ) red deer and dried (Chinese Standards, 2002). Many kinds of amino acids, minerals, sugars, etc. are included (Kim et al., 1973).

Cervus nippons are widely distributed in the northeast (Ohtaishi 1986, Ohtaishi et. Al. 1990, Whitehead 1972, White head 1993), slightly smaller than other deer, weighing 80 kg, 95-180 cm long, 65- 109 cm, with clear buttock spots (Geist 1982). The red deer and elk ( C. canadensis ) have similar appearances, but can distinguish one another clearly. The subspecies of red deer in North America are similar in size to European elk, so they are named North American elk ( Alces alces or moose), which caused confusion of species by attaching another species name 'elk' to red deer. Thus, North American elk was newly named wapiti, and Wapiti and European elk are apparently indistinguishable.

Wapiti and European elk are apparently difficult to distinguish, and the antler from these is also difficult to distinguish. Because deer antler varies considerably in price by region and region, various studies have been attempted to distinguish between them. Karyotyping, Fontana and Rubini (1990), and repetitive DNA analysis (Lima-de Faria et al. , 1984; Bogenberger et al., 1987; Scherthan, 1991), RFLP analysis of mitochondrial DNA (Cronin, 1992), and RNA gene sequencing of mitochondrial DNA (Miyamoto et al., 1990).

Deer antlers are quite different in price depending on the place of origin and site, and most of them are identified in the form of antler, but in the typical state, the form is very similar. Therefore, several methods have been devised to determine the species of deer antler cut from Wapiti, red deer, Japanese deer, roe deer, Capreolus capreolus , as well as Wapiti and European elk. Polziehn and Strobeck, 1998), stellar discrimination using the external forms of 12 antler medicinal herbs (Daixian et al, 1999), and methods using variations of microsatellite polymorphism are known (Poetsch et al., 2001).

Among the various methods, species identification using PCR is one of the most widely used methods because the process is simple and accurate. Among them, multiplex-PCR can be analyzed quickly by preparing species-specific primers and analyzing several samples at the same time (Dean et al., 2005).

Mitochondrial DNA (mt DNA) is suitable as a template to be used as a template for PCR, which has a higher polymorphism since the base substitution rate of mt DNA is faster than that of nuclear DNA (Upholt and Dawid, 1977, Brown et. Al. 1979) . Many variations in species have been reported in many mammals, including humans (Potter et. Al. 1975, Brown et. Al. 1982, Lansman et. Al. 1983). The D-loop present in mtDNA is a hypervariable region, and the base substitution rate is five times higher than that of other mtDNAs (Aquadro et. al. 1983) because it is easy to observe genetic diversity in individuals of various species (Bernatchez et. al. . 1992, Giuffra et. al. 1994, Randi et. al. 1994, Richard et. al. 1996, Wilkinson et. al. 1991, Yang et. al. 1994).

Traditionally, PCR-RFLP (Murray et. Al. 1995) is widely known as a method of identifying antler species, but because of the long process and time-consuming procedure, a method using a species-specific probe (Li et al, 2005) , Sequencing methods (Polziehan and Strobeck, 1998), microsatellite polymorphism (Poetsch et. Al. 2001), RAPD assay (Hidetoshi et al., 1995, Perez et al., 1998) are known, but quickly and accurately identify antler The PCR method is considered to be a consistent and reliable method for this. In particular, the multiplex PCR method, which can identify several species by one PCR, is attracting attention as a very effective method because it can reduce time and money, and improve accuracy and specificity (Dean et al., 2005).

Republic of Korea Patent 1998-0014747 describes a pathogenic E. coli detection method using a multiplex PCR technique.

Korean Patent 1997-002483 describes a method for diagnosing Vibrio infection by PCR using specific oligonucleotide primers.

Korean Patent 1996-0057885 describes a method for determining the origin of Korean ginseng using primer amplification.

As described above, there are many methods for detecting diseases or individuals by using gene-specific primers, but none have been introduced into the selection of deer antler varieties.

Accordingly, the present inventors have identified the present invention by confirming that the antler variety can be distinguished simply and accurately from the antler mixed sample by the multiplex-PCR method using primers specific for the red deer, the Japanese deer, the elk and the reindeer of the present invention. Completed.

An object of the present invention is to provide a primer that specifically binds to the genes of red deer, Japanese deer, elk and reindeer.

In addition, an object of the present invention is to provide a method for identifying antler varieties using the primer.

In order to achieve the above object,

The present invention provides a primer that specifically binds to the D-loop of mitochondrial genes of red deer, Japanese deer, elk and reindeer.

In addition, the present invention provides a method for identifying a antler variety using the primer.

Hereinafter, the present invention will be described in detail.

The present invention provides primers that specifically bind to the genes of red deer, Japanese deer, elk and reindeer.

In the multiplex-PCR reaction that selects the species and reindeer of deer by mixing primers for genes of various types (red deer, Japanese deer, elk, reindeer) and performing a single PCR at the same time as in the present invention, More stringent conditions are required than in PCR alone, and the gene products must be of different sizes to distinguish the gene products amplified on the gel after completion of the reaction. In setting reaction conditions, it is more difficult to set conditions than a single PCR because common reaction conditions for all of a plurality of pairs of primers should be set.

The present inventors proceeded as follows to produce a primer that satisfies the above conditions.

First, in order to amplify the D-loop region of mitochondrial DNA in the whole DNA isolated from red deer, Japanese deer, elk and reindeer, Polziehn et al. CST2 primer (SEQ ID NO: 1) and CST39 primer (SEQ ID NO: 2) reported in (1998) were used.

As a result of amplifying the entire D-loop using the primers, PCR products corresponding to the respective D-loops of red deer (1134 bp), Japanese deer (1,288 bp), elk (1,209 bp), and reindeer (1,142 bp) were obtained. It was. D-loop nucleotide sequence (http://www.ncbi.nlm.nih.gov) of the existing deer species and the antagonistic sequence of the antler base sequence of the D-loop site isolated in this study were compared. As a result of confirming the accuracy, all showed 95% or more similarity, it was confirmed that the D-loop amplified by PCR in the present invention is the D-loop sequence of each species (see FIGS. 1 and 2).

Since the standard D-loop sequence of each species was determined, primers were prepared for PCR for species selection. The forward primer is a common nucleotide sequence of D-loop of red deer, Japanese deer, elk, and reindeer, and has a primer of 18 to 32 mer selected from the nucleotide sequence shown in SEQ ID NO: 15, preferably described in SEQ ID NO: 4. It is a primer. Reverse primers were prepared from the species-specific sequence and red deer, Japanese deer. Reverse primers specific for elk and reindeer are 18 to 32 mer reverse primers selected from the complementary sequences of the nucleotide sequences set forth in SEQ ID NOs: 16, 17, respectively, within the complementary sequences of the nucleotide sequences set forth in SEQ ID NO: 18; 18 to 20 mer selected, and 18 to 23 mer selected from the complementary sequences of the nucleotide sequences set forth in SEQ ID NO: 19, preferably the primers set forth in SEQ ID NOs: 5, 6, 7, and 8, respectively. Each reverse primer was named RED for red deer, NIP for Japanese deer, ELK for elk, and REIN for reindeer. Reindeer are a species of Odocoilinae, a species completely different from Japanese deer, red deer and elk (Geist 1982), but the horns of Rangifer tarnadus are distributed illegally as antler species. Reindeer specific primers were also made for reindeer identification. As an internal control, the L14724 primer and the H15149 primer were prepared to obtain a constant 489bp amplification product from the cytochrome b gene of mitochondrial DNA.

PCR was performed using the primers prepared above for each deer species DNA. As expected, the PCR products of red deer 199bp, Japanese deer 300bp, elk deer 245bp and reindeer 375bp were confirmed. On the other hand, mice, rats, cats, cows, dogs, and humans can only confirm the amplification products of 489bp (cytochrome b), and the bands expected only in antler were identified (see FIG. 3). Therefore, it was confirmed that the primer of the present invention is a primer specific for the D-loop of mitochondrial genes of red deer, Japanese deer, elk and reindeer.

In addition, the present invention

1) extracting DNA from the subject;

2) Amplification of genes specific to each species using primers common to D-loops of red deer, Japanese deer, elk and reindeer genes and primers specific to each species using the DNA as a template for PCR Performing flex-PCR;

3) performing electrophoresis; And

4) Provides a method for identifying antler cultivar consisting of identifying PCR products.

The inventors of the present invention, using the primer of the present invention, confirmed whether the species of the deer can be identified in the mixed sample. First, the whole DNA of each variety is mixed in a 1: 1 ratio, and there are forward primers common to D-loop, reverse primers specific to red deer, Japanese deer, elk and reindeer, and primer pairs for cytochrome amplification as an internal control. The mixture was subjected to multiplex-PCR. As a result, even when using several primers at the same time, it was confirmed that the amplification products in the state of mixing two to four antler samples can also be identified simultaneously without interfering with each other (see Fig. 5).

In order to confirm the possibility of species identification in the actual dry antler, DNA extraction was performed on commercial antler and multiplex-PCR was performed with the primer of the present invention. As a result, it was confirmed that specific products of the antler cultivars are accurately detected without interfering with each other even in various kinds of mixed antler samples (see FIG. 6). Therefore, the multiplex-PCR using the primer of the present invention confirmed that the antler species can be accurately identified even in mixed samples.

The present invention also provides a multiplex kit for identifying a antler variety comprising a primer specifically binding to a gene of a red deer, a Japanese deer, an elk or a reindeer and a primer commonly binding to a D-loop of a mitochondrial gene.

The kit may additionally include reagents for transcription, amplification and product detection and instructions therefor. For example, the kit may contain transcriptases, deoxynucleotides, thermostable polymerases suitable for DNA amplification reactions, and reagents for labeling and detecting nucleic acids.

Since the kit can identify four kinds of antler varieties from the antler sample at the same time, it is possible to find out the varieties and authenticity of the antler in a short time with a small sample.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Deer Antler Sample

Blood samples of red deer, Japanese deer, elk, and reindeer are collected and used in Seoul Grand Park as the standard sample of antler. Other blood include red deer, Japanese deer, and elk, which are raised in private farms in Korea. And dried antlers distributed in Korea and used for the experiment. Whole DNA isolated from the blood or tissue of the antler was extracted using a QIAamp DNA microkit (QIAGEN, Germany) according to user instructions.

<Example 2> Determination of the D-loop partial nucleotide sequence of the deer antler and determination of the standard D-loop nucleotide sequence

Three primers are used for sequencing by amplifying the D-loop sites of red deer ( Cervus elaphus ), Japanese deer ( Cervus nippon ), elk ( Cervus elaphus canadensis ), and Rangifer tarandus mitochondrial DNA (mtDNA). It was. CST2 primers and CST39 primers set forth in SEQ ID NOs: 1 and 2 are described in Polziehn et al. (1998) was used, and the SeqR primer described in SEQ ID NO: 3 was newly synthesized based on the results of sequencing with CST 39 primers because the entire sequence of D-loop could not be sequenced in one experiment. To complete the rest of the sequence analysis, it was obtained the entire sequence.

Sequence analysis of the D-loop site was performed using a Perkin-Elmer DNA terminator Cycle Sequencing Ready Reaction Kit, a DNA amplifier (GeneAmp PCR System 9700, Applied biosystems), and an ABI 310 genetic analyser (Applied biosystems).

Blood and tissue from red deer, Japanese deer, elk and reindeer, CST2 and CST 39 primers were used to amplify the entire D-loop (FIG. 1). PCR reaction conditions, using DNA polymerase (Taq Gold polymerase, Applied biosystems) to denature the primer pair and template described above for 12 minutes at 95 ℃, 1 minute at 94 ℃, 1 minute at 52 ℃ and 72 ℃ The reaction was carried out 30 times for 1 minute at, and extended for 7 minutes at 72 ° C. to terminate the reaction.

The corresponding amplification products 1134bp, 1,288bp, 1,209bp and 1,142bp sized amplification products were sequenced to determine the D-loop standard nucleotide sequence (FIG. 2).

Sequence comparison was performed using ClustalW (ver. 1.75, Thompson et. Al., 1994), using the L14724 primer and H15149 primer shown in SEQ ID NOs: 9 and 10. D-loop base sequence of the registered deer species (http://www.ncbi.nlm.nih.gov) and the antler base of the D-loop site isolated in this study for the production of primers specific to the antler species By comparing the sequences, we confirmed the accuracy of the sequences used. D-loop sequence of red deer showed 98% similarity to genbank (AF016973), and D-loop sequence of Japanese deer showed 95% similarity to genbank (AF016965), and reindeer subspecies ( Rangifer tarandus groenlandicus, AF096413) was recognized as a reindeer, showing 99% similarity to the nucleotide sequence isolated in this experiment. And since the elk base sequence was not registered in the genbank, 99% similarity was compared with the D-loop region sequence isolated from the elk blood bred in Seoul Grand Park and the elk blood bred in domestic farms. This base sequence was determined by the base sequence of the elk.

Example 3 Specimen-Specific Primer Fabrication and Identification

After determining the standard D-loop sequences of red deer, Japanese deer, elk and reindeer, FOR primer (sense; Tm value 58 ° C) (SEQ ID NO: 4) was used as the forward primer, and red deer, Primers (SEQ ID NOS: 5 to 8) that specifically reacted to Japanese deer, elk and reindeer were prepared, and each primer was RED (Tm value 53 ° C), NIP (Tm value 53 ° C), and ELK (Tm value 53 ° C). ) And REIN (Tm value 54 ° C.). For the primer production was used http://www.bioneer.co.kr/tools site.

To obtain species-specific D-loop mtDNA fragments of deer antler, 40 pmole of L14724 (Masuda et. Al. , 1996), H15149 (Masuda et. Al. ,) With 2 pmole of FOR, RED, NIP, ELK, REIN primers and an internal control . 1996) Primer, 10X reaction buffer (Applied biosystems, USA), 1.5 mM MgCl ₂ , 0.2 mM dNTP Mixture, 1.25 units of AmpliTaq Gold polymerase (Applied biosystems, USA) and 1 ng of DNA were added to 25 μl reaction solution.

DNA amplification was performed using GeneAmp PCR System 9700 (Applied biosystems) for 12 minutes at 95 ° C, followed by denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 30 seconds. 30 cycles were repeated and finally extended at 72 ° C. for 7 minutes. 5 μl of amplified product was electrophoresed in 0.5% TBE buffer (45 mM Tris-borate and 1 mM EDTA, pH 8.0) in 2% agarose gel (Amresco) containing 0.5 μg / ml EtBr to confirm amplification results on UV transilluminator. It was.

As a result of PCR with the primers, as expected, amplification products of 489bp and 199bp in red deer, 489bp and 300bp amplification products in Japanese deer, 489bp and 245bp amplification products in Elk deer, and 489bp and 375bp in reindeer. The amplification products of, and can be confirmed in the mouse, rat, cat, cattle, dogs and humans only 489bp amplification products generated from the cytochrome b, the band expected only in antler was confirmed (Fig. 3). Therefore, it was confirmed that the primer of the present invention is specific to antler.

Unlike the previous experiments that identify only one type, the results can be identified simultaneously with the previous results using the primers of RED, NIP, ELK, and REIN (Gao et. Al. 2004, Dean et. Al. 2005, Wan & Fang 2003).

Example 4 Confirmation of Sensitivity of Multiplex PCR Using Species Specific Primer

Effective detection concentration experiments by multiplex-PCR were performed using species identification primers RED, NIP, ELK and REIN. DNA concentrations of standard deer strains were adjusted to 1 ng, 500 pg, 100 pg, 50 pg, 10 pg, 5 pg, 1 pg, and 0.5 pg. As a result, PCR was able to confirm the minimum concentrations found in the four cultivars (FIG. 4). In the case of red deer (A. RED), the amplifiable concentration by the species-specific primer was 5 pg, but the internal control was able to identify up to 50 pg, allowing species identification up to 50 pg total DNA concentration. In the case of Japanese deer (B. NIP), both species-specific primers and internal controls were able to identify species up to 5 pg total DNA concentration. In the case of elk (C. ELK), the amplifiable concentration by the species-specific primer was 10pg, but the internal control was able to identify up to 50pg, it was possible to identify species up to 50pg total DNA concentration. In the case of reindeer (D. REIN), both species-specific primers and internal controls were able to identify up to 5 pg total DNA concentration. Based on these results, it was found that species identification of deer antler could be identified even with at least 50 pg of total DNA.

Example 5 Classification of Antlers Varieties in Mixed Samples Using Multiplex-PCR

This experiment was confirmed whether species identification is possible even in mixed samples. First, 1 ng of total DNA of each cultivar was mixed 1: 1 ng of total DNA for the experiment, and as a result, the amplification of the internal control (489 bp) was not affected. Amplification products in a mixed state up to the antler species were simultaneously identified without interfering with each other (FIG. 5).

In addition, reindeer horns are not the same kind of antler as antler antler, but they are actually distributed like antler, and the minimum detection concentration of mixed reindeer was confirmed to check whether it can be distinguished in sectioned antler. The detectability of red deer and reindeer, Japanese deer and reindeer, and elk and reindeer were confirmed. When the final DNA concentration was adjusted to 1 ng / ul and mixed at various concentrations, when the red deer and the reindeer were mixed, the reindeer was mixed until about 30% of the reindeer was mixed, and the Japanese deer and the reindeer were mixed. In the case of reindeer, the reindeer was mixed until about 10% of the reindeer was found. And when the elk and reindeer were mixed, the reindeer was mixed until the reindeer was mixed about 7% (Fig. 6). Therefore, it was confirmed that mixed species can be identified when at least 30% of other varieties are mixed.

Example 6 Practical Application of Multiplex PCR to Distribution Antlers

1ng of DNA was used for species identification after DNA extraction for commercial antler in order to confirm the possibility of species identification in the actual distribution of dried antler (FIG. 6). As a result, domestic elk (Fig. 7, Lane 1), Russia (Fig. 7, Lane 3), commercially available Russia (Fig. 7, Lane 8) and Canada (Alk, Fig. 7, Lane 9) 489 bp and 245 bp PCR amplification products were confirmed and confirmed as elk, two species of New Zealand produced (Fig. 7, Lane 2 and Lane 7), domestic imported antler (Korean Medical Association, Fig. 7, Lane 4) and domestic imports Deer antlers distributed in antler (Hanhan distribution, Fig. 7, lane 5) was confirmed by the PCR amplification products of 489bp and 199bp red deer antler. In case of imported reindeer, 489 bp and 375 bp PCR amplification products were identified. In the three Chinese varieties (Fig. 7, lanes 6, 10, and 11), 489 bp PCR amplification products were identified as only amplification products of the internal control reindeer. It was confirmed that is not. Therefore, it was confirmed that varieties of antler antler available in the market by the multiplex-PCR method using the primer of the present invention can be selected.

As described above, the primers specific to the Japanese deer, red deer, elk and reindeer of the present invention and the method of identifying antler varieties using the primers are multiplex-PCR methods using primers specific to each species. It is possible to obtain amplification products specific to antler cultivars at the same time with a single PCR, and it is a new method that reacts quickly and accurately and specifically. Therefore, it can be used to determine antler varieties more effectively than the PCR method using only one type of primer pair. Can be.

<110> Korea Institute of Oriental Medicine <120> The primers specific to Cervus elaphus, C. nippon, C. canadensis          and Rangifer tarandus gene and the method to identify Cervi          Parvum cornu species <160> 19 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> CST2 primer <400> 1 taatatactg gtcttgtaaa cc 22 <210> 2 <211> 24 <212> DNA <213> CST39 primer <400> 2 gggtcggaag gctgggacca aacc 24 <210> 3 <211> 20 <212> DNA <213> SeqR primer <400> 3 atgtcctgtg accattgact 20 <210> 4 <211> 21 <212> DNA <213> FOR primer <400> 4 ccctaagact caaggaagaa g 21 <210> 5 <211> 20 <212> DNA <213> RED <400> 5 gtggttggtg gatgtaaaat 20 <210> 6 <211> 20 <212> DNA <213> NIP <400> 6 tatcttacgc accggtttat 20 <210> 7 <211> 20 <212> DNA <213> ELK <400> 7 ttttatgtac tacgagcgca 20 <210> 8 <211> 21 <212> DNA <213> REIN <400> 8 gtgccatgta cgatcaataa t 21 <210> 9 <211> 28 <212> DNA <213> L14724 <400> 9 cgaagcttga tatgaaaaac catcgttg 28 <210> 10 <211> 35 <212> DNA <213> H15149 <400> 10 aaactgcagc ccctcagaaa tgatatttgt cctca 35 <210> 11 <211> 199 <212> DNA <213> C. elaphus <400> 11 ccctaagact caaggaagaa gccatagccc cactatcaac acccaaagct gaagttctat 60 ttaaactatt ccctgatgct tattaatata gttccataaa aatcaagaac tttatcagta 120 ttaaatttcc aaaaagtttt aatatttcaa tacagctttc cactcaacac ccattttaca 180 ttttacatcc accaaccac 199 <210> 12 <211> 299 <212> DNA C. nippon <400> 12 ccctaagact caaggaagaa gccatagccc cactatcaac acccaaagct gaagttctat 60 ttaaactatt ctctgacgct tattaatata gttccataaa aatcaagaac tttatcagta 120 ttaaatttcc aaaaaatttt aatattttaa tacagttttc tactcaacac ccaatttaca 180 tttatgtcct actaattaca caacaaaaca cgtgatataa ccttatgcac ttgtagcaca 240 taaaattaat gcgttaagac ataccatgta caacagcaca taaaccggtg cgtaagata 299 <210> 13 <211> 245 <212> DNA C. e. canadensis <400> 13 ccctaagact caaggaagaa gccatagccc cactatcaac acccaaagct gaagttctat 60 ttaaactatt ccctgacgct tattaatata gttccataaa aatcaagaac tttatcagta 120 ttaaatttcc aaaaaattta atattttaat acagctttct actcaacatc caatttacat 180 tttatgtcct actaattaca cagcaaaaca cgtgatataa ccttatgcgc tcgtagtaca 240 taaaa 245 <210> 14 <211> 375 <212> DNA <213> R. tarandus <400> 14 ccctaagact caaggaagaa gctatagccc cactatcaac acccaaagct gaagttctaa 60 ttaaactatt ccctggcgta tattaatata gctccacaaa attcaagagc cttgtcagta 120 ttaaatttct aaaaaccttc aagaatttaa tacagttctg cactcaatag ccatattata 180 tattaaatat cattaactac ataaatattt tataaacgta catatatggt cctgtacggc 240 tatagtacat aaaattaatg tattaagaca tattatgtat aatagtacat taaattatat 300 gccccatgct tataagcaag tacttgacat tatttacagt acatagtaca taatattatt 360 gatcgtacat ggcac 375 <210> 15 <211> 80 <212> DNA <213> conserved sequence <400> 15 acctccctaa gactcaagga agaagccata gccccactat caacacccaa agctgaagtt 60 ctatttaaac tattctctga 80 <210> 16 <211> 49 <212> DNA <213> C. elaphus primer region <400> 16 tacattttac atccaccaac cacacaacaa aatatgtaat aaaacctta 49 <210> 17 <211> 77 <212> DNA <213> C.nippon primer region <400> 17 atgtacaaca gcacataaac cggtgcgtaa gatacattat gcatgatagt acataaatta 60 atgtattagg acatact 77 <210> 18 <211> 20 <212> DNA C. e. canadensis primer region <400> 18 tgcgctcgta gtacataaaa 20 <210> 19 <211> 23 <212> DNA <213> R.tarandus primer region <400> 19 atattattga tcgtacatgg cac 23  

Claims (21)

Red deer ( Cervus elaphus ) A reverse primer that specifically binds to the D-loop of the mitochondrial gene of. The reverse primer of claim 1, wherein the primer is selected from the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 16. The reverse primer according to claim 2, wherein the primer has a nucleotide sequence set forth in SEQ ID NO: 5. A primer that specifically binds to the D-loop of the mitochondrial gene of Japanese deer ( C. nippon ). The reverse primer of claim 4, wherein the primer is selected from the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 17. 6. The reverse primer according to claim 5, wherein the primer has a nucleotide sequence set forth in SEQ ID NO: 6. A primer that specifically binds to the D-loop of the mitochondrial gene of Elk ( C. canadensis ). The reverse primer of claim 7, wherein the primer is selected from the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 18. 9. The reverse primer according to claim 8, wherein the primer has a nucleotide sequence set forth in SEQ ID NO: 7. A primer that specifically binds to the D-loop of the mitochondrial gene of Reindeer ( Rangifer tarandus ). The reverse primer of claim 10, wherein the primer is selected from the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 19. 12. 12. The reverse primer of claim 11, wherein the primer has a nucleotide sequence set forth in SEQ ID NO: 8. Forward primer that commonly binds to the D-loop of mitochondrial genes of Japanese deer, red deer, elk and reindeer. 14. The primer of claim 13, wherein the primer is selected from the nucleotide sequences set forth in SEQ ID NO: 15. 15. The forward primer of claim 14, wherein the primer has a nucleotide sequence set forth in SEQ ID NO: 4. 1) extracting DNA from the subject; 2) Multiplex-PCR amplifying genes specific to red deer, Japanese deer, elk or reindeer using the DNA extracted in step 1) as a template and using the primers of claim 13 and primers 1, 4, 7, and 10 Performing; 3) performing electrophoresis; And 4) identifying the PCR product; antler variety identification method consisting of. 17. The method according to claim 16, wherein the gene specific for the red deer of step 2) is set forth in SEQ ID NO: 11. 17. The method according to claim 16, wherein the gene specific for the Japanese deer of step 2) is set forth in SEQ ID NO: 12. 17. The method according to claim 16, wherein the gene specific for the elk of step 2) is set forth in SEQ ID NO: 13. 17. The method of claim 16, wherein the gene specific for the reindeer of step 2) is described in SEQ ID NO: 14. Multiplex kit for identifying antler varieties comprising the primers of claim 1, 4, 7, 10 and 13.

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