KR102055308B1 - Composition for identifying place of origin of deer antlers and method for identifying the type of deer antlers comprising the same - Google Patents

Abstract

It relates to a composition for determining the deer antler origin and a method for determining the type of deer antler comprising the same. According to a composition according to one aspect and a method of determining using the same, the present invention minimizes non-specific annealing that may be caused by a conventional method of determining the origin of antler by comparing the type of fluorescence and the temperature at which the intensity of fluorescence rapidly decreases. The specificity of the discrimination could be improved. Therefore, the present invention can quickly and specifically determine New Zealand deer antler, Chinese deer antler, Russian deer antler, or elk deer antler.

Description

Composition for determining the origin of deer antler and a method for discriminating the deer antler comprising the same {Composition for identifying place of origin of deer antlers and method for identifying the type of deer antlers comprising the same}

It relates to a composition for determining the deer antler origin and a method for determining the species of antler including the same.

Deer antler is a deer's young horn, which is cut and dried from the horn of a soft deer that has not yet been ossified or slightly ossified. The substrate of the antler is soft and soft, the surface of the antler is covered with soft fur, and the inner surface of the antler is filled with the blood of a deer. According to Dongbogam and herbaceous tree, antler is known to boost the body's energy, enhance brain function, and strengthen bones and muscles.

Recently, the demand for finding antler for health is increasing, but since the difference in efficacy is remarkable according to the origin of the antler and the part of the contained antler, special attention is required. Currently, the antlers distributed in Korea include Chinese antler, New Zealand antler, and Russian antler. Among them, the Russian antler is a deer antler that has grown over the cold altitudes of 2000m above sea level and 30 ℃ below zero.元) is known as 'Won Yong' and has the reputation of the world's best quality. Canadian deer antler, on the other hand, has been banned since 2000 due to the melanoma disease of Canadian deer ( C. elaphus canadensis , ELK). However, some Canadian antlers, that is, elk antlers, are distributed through wheat trade through China, and in some cases they are mixed with Russian dragons, causing social problems. Despite the difference in efficacy and quality according to the antler origin, it is almost impossible to determine the origin of the antler by visually dividing it with the naked eye.

Under these technical backgrounds, various studies have been conducted on techniques for determining the origin of antler. As one of these techniques, Korean Patent Publication No. 10-2009-0015587 analyzes the variable sequences of the genes of Russian deer ( Cervus elaphus sibiricus ), Chinese deer ( Cervus elaphus bartrianus ), New Zealand deer, and Canadian deer ( C. elaphus canadensis , ELK). Oligonucleotides for screening deer breeds, and antler breed discrimination methods using the oligonucleotides are disclosed, but such techniques may cause nonspecific annealing, and due to their low sensitivity, the effectiveness of the determination of antler origin is very high. There is a limit of low.

One aspect is to provide a composition for determining the origin of antler, including a primer set and a probe for specifically discriminating New Zealand antler, Chinese antler, Russian antler, or elk antler.

Another aspect is to provide a kit for determining a deer antler origin comprising the composition.

Another aspect includes (a) preparing a reaction mixture comprising antler sample DNA and the composition; (b) performing a PCR reaction on the antler sample DNA and inducing binding between the PCR amplification product and the probe; And (c) confirming binding between the PCR amplification product and the probe through analysis of a fluorescence fusion curve according to temperature change.

One aspect includes (a) a first primer pair for amplifying the 112th to 135th base sequence of SEQ ID NO: 1 or the 134th to 156th base sequence of SEQ ID NO: 2, and the 564th base of SEQ ID NO: 3 A primer set comprising any one or more primer pairs selected from the second primer pair for amplifying the 581 nucleotide sequence from the sequence or the 681 nucleotide sequence from the 681 nucleotide sequence of SEQ ID NO: 4; And

(b) a first probe consisting of the nucleotide sequence of SEQ ID NO: 9 for discriminating New Zealand antler, a second probe consisting of the nucleotide sequence of SEQ ID NO: 10 for discriminating Chinese antler, SEQ ID NO: 11 for discriminating Russian antler It provides a composition for determining the origin of antler, comprising a third probe consisting of the nucleotide sequence of any one or more probes selected from the fourth probe consisting of SEQ ID NO: 12 for determining the elk antler.

As used herein, the term "primer" will serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable temperature and buffer. Single-stranded oligonucleotides. In addition, "forward primer" and "reverse primer" means primers that bind to the 3 'end and 5' end of a predetermined portion of the template to be amplified by polymerase chain reaction, respectively.

As used herein, the term "probe" refers to a natural or modified monomer or linear oligomer with bonds, including deoxyribonucleotides that can hybridize to a specific polynucleotide sequence.

Detergent composition for determining the origin of antler according to an embodiment includes a primer set and a probe for targeting the nucleotide sequence of the mitochondrial D-loop region, through the combination of New Zealand antler, Chinese antler, Russian antler, or elk antler Can be specifically detected and discriminated.

The first primer pair is for amplifying the nucleotide sequence in the mitochondrial D-loop region of New Zealand deer antler and / or Chinese deer antler, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 5 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 6 It may be made of. The second primer pair is for amplifying the nucleotide sequence in the mitochondrial D-loop region of Russian antler and / or elk antler, and includes a forward primer consisting of the nucleotide sequence of SEQ ID NO: 7 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 8 It may be made of. If the primer pair is capable of amplifying the nucleotide sequence as described above, each having at least 80%, at least 85%, at least 90%, at least 95%, and at least 95% sequence identity with the nucleotide sequences of SEQ ID NOs: 5 to 8, respectively. It may be composed of a nucleotide sequence.

The probe may detect a target sequence for determination of a deer antler origin from a PCR amplification product through the primer pair. The probe may be labeled with a fluorescent material. 6-FAM (6-carboxyfluorescein), TET (2 ', 7'-dichloro-6-carboxy-4,7-dichlorofluorescein), Texas Red, 6-JOE (6-carboxy-4', 5) '-dichloro-2', 7'-dimethoxyfluorescein), HEX (2 ', 4', 5 ', 7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), Cy3 (Cyanine 3), Cy5, rhodamine (Rhodamine) and VIC may be applied, but is not limited thereto.

In addition, the probe may be labeled with a fluorescent material at the 5 'end and a matting material at the 3' end for visualization using the Fluorescent Resonance Energy Transfer (FRET) principle. As the matting material, 6-TAMRA (6-carboxytetramethylrhodamine), BHQ (Black Hole Quencher) -1, BHQ-2, and BHQ-3 may be used, but the present invention is not limited thereto.

In one embodiment, the first to fourth probes can be independently labeled with a fluorescent material having a different wavelength band at each 5 'end for determination of the antler origin. Since phosphors with inherent excitation and emission wavelengths are labeled differently for each probe, the binding between the probe and the target sequence can be confirmed by detecting the inherent signal from the phosphor. . For example, 6-FAM may be labeled at the 5 'end of the first probe for determining New Zealand antler, and HEX may be labeled at the 5' end of the second probe for determining Chinese antler. In addition, Texas Red may be labeled at the 5 'end of the third probe for determining Russian antler, and Cy5 may be labeled at the 5' end of the fourth probe for determining elk antler.

In one embodiment, the composition comprises a primer set comprising a first primer pair for amplifying the 112th base sequence from SEQ ID NO: 1 to 135 base sequence and 134th base sequence from 156 base sequence of SEQ ID NO: 2; And a combination of a first probe consisting of a nucleotide sequence of SEQ ID NO: 9, and / or a second probe consisting of a nucleotide sequence of SEQ ID NO: 10, wherein the combination detects and discriminates New Zealand antler and / or Chinese antler. can do.

In one embodiment, the composition comprises a primer set comprising a second primer pair for amplifying the 564th base sequence from 581 base sequence of SEQ ID NO: 3 and the 681th base sequence from 700th base sequence of SEQ ID NO: 4; And a third probe consisting of a nucleotide sequence of SEQ ID NO: 11, and / or a fourth probe consisting of SEQ ID NO: 12, through which the Russian antler and / or elk antler may be detected and determined. have.

Another aspect provides a kit for determining a deer antler origin comprising the composition.

In the antler origin determination kit, the same terms or elements mentioned above are the same as those mentioned above.

The antler origin determination kit according to an embodiment may further include tools or reagents necessary for PCR reaction or PCR amplification product detection in addition to the above-described primer set or probe. For reagents, for example, DNA polymerase, dNTP mixtures (dATP, dCTP, dGTP, dTTP), PCR buffers and DNA polymerase cofactors may be included separately.

Another aspect includes (a) preparing a reaction mixture comprising antler sample DNA, primer sets, and probes; (b) performing a PCR reaction on the antler sample DNA and inducing binding between the PCR amplification product and the probe; And (c) confirming the binding between the PCR amplification product and the probe through analysis of fluorescence labeled on the probe,

The primer set includes a first primer pair for amplifying the base sequence from SEQ ID NO: 1, 112 through 135, or the sequence 134 through SEQ ID NO: 156, and 564 from SEQ ID NO: 3 Any one selected from the group consisting of at least one primer pair selected from a 581 nucleotide sequence or a second primer pair for amplifying the 700 th nucleotide sequence from the 681 nucleotide sequence of SEQ ID NO: 4,

The probe may comprise a first probe consisting of a nucleotide sequence of SEQ ID NO: 9 for determining a New Zealand antler, a second probe consisting of a nucleotide sequence of SEQ ID NO: 10 for a Chinese antler, and a SEQ ID NO: 11 for determining a Russian antler At least one selected from a third probe consisting of a nucleotide sequence of 4, and a fourth probe consisting of SEQ ID NO: 12 for determining elk antler, wherein the first to fourth probes have a fluorescent material at a 5 'end. Provided is a method of determining the origin of antler, which is labeled and labeled with a quencher at the 3 ′ end.

In the method of determining the antler origin of origin, the same terms or elements mentioned above are the same as those mentioned above.

According to one embodiment, the method comprises amplifying a nucleotide sequence in the mitochondrial D-loop region from the antler sample DNA through a PCR reaction using the primer set described above; And annealing the target sequence and the probe in the PCR amplification product. The PCR reaction and annealing conditions can be carried out under conditions known in the art, which can be changed in any way.

The binding between the target sequence and the probe through a series of reactions can be confirmed by a specific signal in the wavelength range derived from the fluorescent material labeled at the 5 'end of the probe. Since each probe is labeled with a different fluorescent substance, it is possible to easily discriminate between New Zealand deer antler, Chinese deer antler, Russian deer antler, or elk deer antler by detecting a unique signal derived from the phosphor.

In addition, the analysis of the fluorescence may include performing a fluorescence melting curve analysis (Fluorescence melting curve analysis) while increasing the temperature step by step.

The fluorescence fusion curve analysis, for example, while increasing the temperature from about 40 ℃ to about 80 ℃ by about 0.5 ℃, the temperature at which the bond between the target sequence and the probe is dissociated, that is, the intensity of fluorescence is rapidly reduced Measuring the temperature (Melting temperature: Tm) may be included. The assay can be performed using methods known in the art, for example using the Bio-Rad CFX96 instrument.

As a result of the fluorescence melting curve analysis, when the Tm is 59.5 ° C., it can be determined as New Zealand deer antler, and when the Tm is 63 ° C., it can be determined as Chinese deer antler, and when the Tm is 63.5 ° C., Russian deer antler or Can be determined by Elk Antler.

In one embodiment, the reaction mixture comprises a primer set comprising a first primer pair for amplifying the 112th base sequence from SEQ ID NO: 1 to 135 base sequence and the 134th base sequence from 134 base sequence of SEQ ID NO: 2; And a combination of a first probe consisting of a nucleotide sequence of SEQ ID NO: 9, and / or a second probe consisting of a nucleotide sequence of SEQ ID NO: 10, through which a New Zealand antler and / or Chinese antler can be determined. have.

In one embodiment, the reaction mixture comprises a primer set comprising a second primer pair for amplifying the 564th base sequence from 581 base sequence of SEQ ID NO: 3 and the 681th base sequence from 700 base sequence of SEQ ID NO: 4; And a third probe consisting of a nucleotide sequence of SEQ ID NO: 11, and / or a fourth probe consisting of SEQ ID NO: 12, through which the Russian antler and / or elk antler can be determined.

According to one embodiment, the individual discrimination of the two kinds of compositions having the aforementioned combinations can minimize non-specific annealing that can result from conventional discrimination methods, thereby remarkably reducing the specificity of the antler origin determination. Could improve.

According to the composition according to one aspect and a method for determining using the same, a small amount of antler samples can be used to quickly and specifically determine New Zealand antler, Chinese antler, Russian antler, or elk antler.

According to the composition of the present invention and a method of determining using the same, non-specific annealing that can be caused by the conventional method of minimization is minimized, and antler is compared by comparing the type of fluorescence and the temperature at which the intensity of fluorescence rapidly decreases. The specificity of origin determination can be further improved.

Figure 1 shows the site in the mitochondrial D-loop region of New Zealand deer antler specifically amplified by the first primer pair.
Figure 2 shows the site in the mitochondrial D-loop region of Chinese antler specifically amplified by the first primer pair.
Figure 3 shows the site in the mitochondrial D-loop region of the Russian deer antler specifically amplified by the second primer pair.
Figure 4 shows the sites in the mitochondrial D-loop region of elk antler specifically amplified by a second primer pair.
Figure 5 shows the target sequence in the mitochondrial D-loop region of New Zealand antler, Chinese antler, Russian antler, elk antler.
Figure 6 shows an example of a determination test of the antler origin according to one embodiment.
Figure 7 shows the change in temperature and reaction time for asymmetric real-time PCR and fluorescence melting curve analysis in the determination method of antler origin according to an embodiment.
8 is a result confirmed by the fluorescence melting curve analysis of the experimental results according to the determination method of the antler origin according to an embodiment.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Primer Set for Determination of Antler Origin

In this example, a primer set for determining a deer antler origin was produced based on gene information of NCB GeneBank and mitochondrial D-loop region (Region). Deer Antlers of Russian Deer (hereinafter referred to as Russian Deer Antlers), Deer Antlers of Chinese Deer (hereinafter referred to as 'Chinese Deer Antlers'), Deer Antlers of New Zealand Deer Antlers (hereinafter referred to as 'New Zealand Deer Antlers') and Elk For each DNA extracted from the antler sample, the primer was designed to amplify a specific region of the gene of the mitochondrial D-loop region.

Specifically, in order to improve the specificity of the detection in the amplification process by the polymerase chain reaction (PCR) reaction, the primer set may contain DNA of mitochondrial D-loop region derived from New Zealand deer antler and Chinese deer antler. A first primer pair capable of specifically amplifying and a second primer pair capable of specifically amplifying the DNA of the mitochondrial D-loop region derived from Russian antler and elk antler were designed.

1 and 2 show sites in the mitochondrial D-loop region of New Zealand deer antler and Chinese deer antler specifically amplified by the first primer pair.

3 and 4 show regions in the mitochondrial D-loop region of Russian antler and elk antler specifically amplified by a second primer pair. In Figures 1 to 4, the site amplified by the PCR reaction is yellow, the site where the primer is bound is red, the site where the probe is bound is underlined.

The base sequence and the size of the amplification product of the primer set according to one embodiment for determining the antler origin are as shown in Table 1 and Table 2.

Example 2 Preparation of Determination of Deer Antler Origin

In this embodiment, a antagonist origin determination probe for the detection of each target sequence was prepared based on gene information of NCB GeneBank and mitochondrial D-loop region (Region). Specifically, New Zealand deer antler, Chinese deer antler, Russian deer antler, or elk deer antler was prepared to have a sequence 100% complementary to the target sequence in the mitochondrial D-loop region.

Figure 5 shows the target sequence in the mitochondrial D-loop region of New Zealand antler, Chinese antler, Russian antler and elk antler. In FIG. 5, each target sequence is indicated by a red box, and the target sequence of the mitochondrial D-loop gene of New Zealand deer antler is designed differently from the target sequence of Chinese deer antler 8bp, and the D-loop gene of Russian deer antler The target sequence was designed 2bp different from the target sequence of the elk antler.

The base sequence of the probe according to one embodiment for determining the antler origin is as shown in Table 3 below.

In addition, in order to visualize using the fluorescence resonance energy transfer principle, the probe according to the embodiment is labeled with a fluorescent material and a quencher at both ends. The double labeled probe was synthesized by an integrated DNA Technologies, Inc. company. That is, when the probe having the following structure is bound to the target sequence of the antler DNA sample, the probe becomes a straight line according to this binding, and thus the distance between the fluorescent material labeled on both ends and the quencher is farther away, causing fluorescence. It is designed to be. On the contrary, when the probe having the following structure is separated from the target sequence of the antler DNA sample by the temperature rise, the probe is converted into the conventional twisted structure, and thus the fluorescent substance labeled at both ends and It is designed to rapidly reduce the fluorescence as the distance between the matting materials is close.

The structure of the double-labeled probe according to one embodiment for determining the antler origin is as shown in Table 4 below.

Example 3 Determination of Deer Antler Origin Using Fluorescence Fusion Curve Analysis

3-1. Determination process of Deer Antler origin

Figure 6 shows an example of a determination test of the antler origin according to one embodiment. In FIG. 6, Reaction 1 is used to discriminate between New Zealand antler and Chinese antler, a New Zealand type probe labeled with 6-FAM at the first primer pair and 5 'end, and a Chinese type probe labeled at 5' end with HEX. It was intended to induce a specific reaction with the sample DNA in the reaction mixture added. Reaction 2 is for discriminating Russian deer antler and elk deer antler, a reaction comprising a second primer pair, a Russian type probe labeled 5 'end with Texas Red, and an Elk type probe labeled 5' end with Cy5 It was intended to elicit specific reactions with sample DNA in the mixture.

(1) Preparation of Sample DNA

Samples were prepared in powder form by crushing 1 cm (horizontal) x 1 cm (vertical) antler slices into a bowl. Meanwhile, the samples were prepared from New Zealand deer antler, Chinese deer antler, Russian deer antler, and elk deer antler, respectively, to obtain sample DNA through the following procedure.

Samples prepared in powder form, 300 μl of Buffer CL, and 20 μl of proteinase K were added to GeneAll's Exgene ^™ Genomic DNA micro kit, which was vortexed and incubated at 56 ° C. for 30 minutes. Thereafter, the incubated mixed solution was spun down, and 300 μl of Buffer BL was added thereto, and then again incubated at 70 ° C. for 10 minutes. The incubated mixed solution was centrifuged at 13,000 rpm for 1 minute and the supernatant obtained therefrom was transferred to a 1.5 ml tube. 300 μl of 100% ethanol was added to the tube followed by vortexing and spin-down treatment. The solution in the tube was transferred to the micro column of the Kit, which was centrifuged for 1 minute at over 8,000 rpm. After replacing the collection tube with a new one, 500 μl of Buffer BW was added to it, followed by centrifugation for 1 minute at 8,000 rpm and higher. Then, 700 μl of Buffer BW was added thereto, followed by centrifugation for 1 minute over 8,000 rpm. Then, the solution in the collection tube was discarded, combined with the column again, and centrifuged at 13,000 rpm for 3 minutes. After the new 1.5ml tube was combined with the column, 50µl of Buffer AE was added to the column and left for 1 minute. Thereafter, this was centrifuged at 13,000 rpm for 1 minute to obtain sample DNA, and the concentration of the obtained DNA was measured, which was then diluted to 10 ng / ul.

(2) Asymmetric Real-Time PCR and Fluorescence Fusion Curve Analysis

Asymmetric real-time PCR reaction and fluorescence fusion curve analysis were performed according to the protocol of the Bio-Rad CFX96 instrument.

The reaction mixture of Reaction 1 was prepared in a composition as shown in Table 5 below, and the reaction mixture was prepared in an amount such that 5 reactions could proceed per sample.

The reaction mixture of Reaction 2 was prepared in a composition as shown in Table 6 below, and the reaction mixture was prepared in an amount such that 5 reactions could proceed per sample.

Then, in the 8-strip tube shown in FIG. 6, 15 μl of the reaction mixture of Reaction 1 was dispensed into tubes 1 to 4, and 15 μl of the reaction mixture of Reaction 2 was dispensed into tubes 5 to 8. Here, 5 μl of sample DNA was added to tubes 1 to 3 and 5 to 7 and 5 μl of ultra pure water was added to tubes 4 and 8 as controls. Tubes 1 to 8 containing the reaction mixture and the like were centrifuged and placed in a Bio-Rad CFX96 instrument to perform plate setting.

Then, asymmetric real-time PCR and fluorescence fusion curve analysis were performed at the temperature and reaction conditions shown in Table 7 and FIG. 7.

3-2. Judgment result of deer deer origin

8 is a result confirmed by the fluorescence melting curve analysis of the experimental results according to the determination method of the antler origin according to an embodiment. In the fluorescence fusion curve, the x axis is temperature (° C.) and the y axis represents the change in fluorescence intensity per unit time.

As a result, as a result corresponding to the origin of the antler sample DNA, the binding of each sample DNA and a probe specific to the antler origin (probe 1-4) results in a specific signal at each corresponding wavelength originating from the labeled fluorescent substance. I could confirm through. That is, in a tube containing the reaction mixture of Reaction 1, the New Zealand antler DNA sample is signaled by 6-FAM labeled at the 5 'end of Probe 1, and the Chinese Antler DNA sample is labeled at the 5' end of Probe 2. The signal by HEX was shown. Also, in the tube containing the reaction mixture of Reaction 2, the Russian antler DNA sample was signaled by Texas Red labeled at the 5 'end of probe 3, and the elk antler DNA sample was labeled at Cy5 labeled at the 5' end of probe 4. Showed a signal by In particular, the intensity of the detected fluorescence was very high, in the range of about 300 to 1000 -d (RFU) / dT, and showed a high level of specificity, so that the origin of antler was more clearly identified through the designed primers and probes. Could be determined.

In addition, while gradually increasing the temperature of the solution in the tube from the starting temperature of 40 ℃ to about 80 ℃, the temperature of the target sequence (Melting temperature: Tm) was measured. As a result, as shown in Fig. 8, in each fluorescence fusion curve, the New Zealand antler DNA sample had a Tm of 59.5 ° C, the Chinese antler DNA sample had a Tm of 63 ° C, the Russian antler DNA sample and the elk antler DNA were 63.5 ° C. Tm is shown. In other words, as a measure to determine the origin of antler, it has been shown that the change in the Tm can also be utilized.

The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> Mediforum co., Ltd <120> Composition for identifying place of origin of deer antlers and          method for identifying the type of deer antlers configuring the          same <130> PN123737KR <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 916 <212> DNA <213> Cervus elaphus mitochondrion D-loop <400> 1 gcttattaat atagttccat aaaaatcaag aactttatca gtattaaatt tccaaaaagt 60 tttaatattt caatacagct ttccactcaa cacccatttt acattttaca tccacctacc 120 acacaacaaa atatgtaata aaaccttatg cgcttatagt acatagaatt aatgtactag 180 gacatactat gtataatagt acattatatt atatgcccca tgcatataag catgtacttt 240 ctattattta tagtacatag tacatgatgt tgttcatcgt acatagcgca ttaagtcaaa 300 tcagtccctg tcaacatgcg tatcccgtcc cctagatcac gagcttgatc accatgccgc 360 gtgaaaccag caacccgctg ggcagggatc cctcttctcg ctccgggccc atgaactgtg 420 ggggtagcta tttaatgaac tttatcagac atctggttct tttttcaggg ccatctcacc 480 taaaatcgcc cactccttgc aatataagac atctcgatgg actaatgact aatcagccca 540 tgctcacaca taactgtggt gtcatacatt tggtattttt aatttttggg gggatgcttg 600 gactcagcaa tgaccgtctg gcggtcccgt cccggagcat gaattgtagc tggacttaac 660 tgcatcttga gcatccccat aatggtaggc atgggcatgg cagtcaatgg tcacaggaca 720 taatcattat ttcatgagtc aaccctaaga tctattttcc cccccctgct aattttttcc 780 cccttatata gttatcacca tttttaacac acttttccct aaatattatt ttaaatttat 840 cacatttcca atactcaaat cagcactcca gggggtggta actatataaa cgccaatttt 900 tccctaatta tgtaca 916 <210> 2 <211> 919 <212> DNA <213> Cervus elaphus bactrianus isolate BAT2 D-loop <400> 2 acgcttatta atatagttcc atagaaatca agaactttat cagtattaaa tttccaaaaa 60 attttaatat ttcaatacag ctttctactc aacatccatt ttacatctta cattacacta 120 accacataac aaaacacata atataaccct atgcgcttat agtacataga attaatgtat 180 taggacatac tatgtataat agtacattat attatatgcc ccatgcttat aagcatgtac 240 tttccattgt ttacagtaca tagtacatga tgttgttcat cgtacatagc gcattaagtc 300 aaatcagttc ttgtcaacat gcgtatcccg tcccctagat cacgagctta actaccatgc 360 cgcgtgaaac cagcaacccg ctgggcaggg atccctcttc tcgctccggg cccatagatc 420 gtgggggtag ctatttaatg aattttacca ggcatctggt tcttttttca gggccatctc 480 atctaaaatc gcccactcct tgcagtataa gacatctcga tggactaatg actaatcagc 540 ccatgctcac acataactgt ggtgtcatac atttggtatt tttaattttt ggggggatgc 600 ttggactcag caatggccgt ctgaggcccc gtctcggagc atgaattgta gctggactta 660 actgcatctt gagcatcccc ataatggtag gcatgggcat ggcagtcaat ggtcacagga 720 cataattatt atttcataaa ccaaccctaa gatctatttc ccccccctcc gctaattttt 780 cccccttata tagttatcac catttttaac acacttttcc ctagatatta ttttaaattt 840 atcacatttc caatactcaa attagcactc cagggggtgg taagtatata aacgccaatt 900 tttccctaat tatgcatag 919 <210> 3 <211> 994 <212> DNA <213> Cervus elaphus sibiricus isolate SIB2 D-loop <400> 3 acgcttatta atatagttcc ataaaaatca agaactttat cagtattaaa tttccaaaaa 60 atttaatatt ttaatacagc tttctactca acatccaatt tacattttat gtcctactaa 120 ttacacagca aaacacatga tataacttta tgcactcgta gtacataaaa tcaatgtgct 180 aggacataca tgtataacag tacatgagtt agcgtatagg acatactatg tataatagta 240 cataaattaa tgtattagga catattatgt ataatagtac attatattat atgccccatg 300 cttataagca tgtacccttc actatctaaa gtacatagta cataatgttg tccatcgtac 360 atagcacatt aagtcaaatc agtccttgtc aacatgcgta tcccgtcccc tagatcacga 420 gcttaattac catgccgcgt gaaaccagca acccgctggg cagggatccc tcttctcgct 480 ccgggcccat ggaccgtggg ggtagctatt taatgaactt tatcagacat ctggttcttt 540 tttcagggcc atctcatcta aaatcgccca ctccttgcaa tataagacat ctcgatggac 600 taatgactaa tcagcccatg ctcacacata actgtggtgt catacatttg gtatttttaa 660 tttttggggg gatgcttgga ctcagcaatg gccgtctgag gccccgtccc ggagcatgaa 720 ttgtagctgg acttaactgc atcttgagca tccccataat ggtaggcgca gggcattaca 780 gtcaatggtc acaggacata attattattt catgagtcaa ccctaagatc tatttccccc 840 cccttcttat tttttccccc ttatatagtt atcaccattt ttaacacact ttcccctaga 900 tataatttta aatttatcac atttccaata ctcaaaatag cactccagag ggaggtaagt 960 atataaacgc caatttttcc ctaattatgc atag 994 <210> 4 <211> 1208 <212> DNA <213> Cervus canadensis nelsoni isolate NEL4 D-loop <400> 4 taatatactg gtcttgtaaa ccagaaaagg agagcaacca acctccctaa gactcaagga 60 agaagccata gccccactat caacacccaa agctgaagtt ctatttaaac tattccctga 120 cgcttattaa tatagttcca taaaaatcaa gaactttatc agtattaaat ttccaaaaaa 180 tttaatattt taatacagct ttctactcaa catccaattt acattttatg tcctactaat 240 tacacagcaa aacacgtgat ataaccttat gcgctcgtag tacataaaat caatgtgcta 300 ggacatgcat gtataacagt acatgagtta gcgtatagga catattatgt ataatagtac 360 ataaattaat gtattaagac atattatgta taatagtaca ttatattata tgccccatgc 420 ttataagcat gtacttctca ctatctgaag tacatagtac ataatgttgt tcatcgtaca 480 tagtacatta agtcaaatca gtccttgtca acatgcgtat cccgtcccct agatcacgag 540 cttaattacc atgccgcgtg aaaccagcaa cccgctgggc agggatccct cttctcgctc 600 cgggcccatg aaccgtgggg gtagctattt aatgaatttt atcagacatc tggttctttt 660 ttcagggcca tctcatctaa aatcgcccac tccttgtaac ataagacatc tcgatggact 720 aatgactaat cagcccatgc tcacacataa ctgtggtgtc atacatttgg tatttttaat 780 ttttgggggg atgcttggac tcagcaatgg ccgtctgagg ccccgtcccg gagcatgaat 840 tgtagctgga cttaactgca tcttgagcat ccccataatg gtaggcgcag ggcattacag 900 tcaatggtca caggacatag ttattatttc atgagtcaac cctaagatct atttcccccc 960 ccttcttatt tttccccctt atatagttat caccattttt aacacacttt cccctagata 1020 taattttaaa tttatcacat ttccaatact caaaatagca ctccagaggg aggtaagtat 1080 ataaacgcca atttttccct aattatgcat agttaatgta gcttaaacag caaagcaagg 1140 cactgaaaat gcctagatga gtatattaac tccataaaca cacaggtttg gtcccagcct 1200 tccgaccc 1208 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer 1 forward <400> 5 caagaacttt atcagtatta aatttc 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer 1 reverse <400> 6 tacattaatt ctatgtacta taagcgc 27 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer 2 forward <400> 7 gaattttatc agacatctgg ttct 24 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 2 reverse <400> 8 attgactgta atgccctgc 19 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe 1 <400> 9 ccacctacca cacaacaaaa tatg 24 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe 2 <400> 10 acacataata taaccctatg cgc 23 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe 3 <400> 11 tcgcccactc cttgcaat 18 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe 4 <400> 12 aatcgcccac tccttgtaac 20

Claims (13)

A composition for determining the origin of antler, comprising a first and a second composition,
The first composition comprises a primer set comprising a first primer pair for amplifying the 112th to 135th base sequence of SEQ ID NO: 1 and the 156th to 156th sequence of SEQ ID NO: 2, and New Zealand deer deer antler. A first probe consisting of a nucleotide sequence of SEQ ID NO: 9 for discriminating, and a second probe consisting of a nucleotide sequence of SEQ ID NO: 10 for discriminating Chinese antler,
The second composition comprises a primer set and a Russian deer antler comprising a second primer pair for amplifying the 564 th base sequence from SEQ ID NO: 3 to the 581 th base sequence and the 681 th base sequence to the 700 th base sequence of SEQ ID NO: 4 Composition comprising a third probe consisting of the nucleotide sequence of SEQ ID NO: 11 for discrimination, and the fourth probe consisting of SEQ ID NO: 12 for discriminating elk antler.
The method of claim 1, wherein the first primer pair is a forward primer consisting of a nucleotide sequence of SEQ ID NO: 5 and a reverse primer consisting of a nucleotide sequence of SEQ ID NO: 6,
The second primer pair is a forward primer consisting of the nucleotide sequence of SEQ ID NO: 7 and the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 8, composition for determining the origin of antler.
The method of claim 2, wherein the first primer pair is for amplifying genes of New Zealand antler and Chinese antler, the second primer pair is for amplifying genes of Russian antler and elk antler,
The gene is derived from the gene of the mitochondrial D-loop region, the composition for determining the deer antler origin.
The composition of claim 1, wherein the first to fourth probes are labeled with a fluorescent material at the 5 ′ end and a quencher at the 3 ′ end.
The composition of claim 4, wherein the fluorescent material labeled at the 5 ′ end of the first to fourth probes is different for each probe.
The composition of claim 4, wherein the composition is used for fluorescence curve analysis.
delete Kit for determining the origin of antler, comprising the composition of any one of claims 1 to 6.
(a) preparing a reaction mixture comprising a composition for detecting antler origin comprising a antler sample DNA, a first and a second composition;
(b) performing a PCR reaction on the antler sample DNA and inducing binding between the PCR amplification product and the probe; And
(c) confirming the binding between the PCR amplification product and the probe through analysis of fluorescence labeled on the probe,
The first composition comprises a primer set comprising a first primer pair for amplifying the 112th to 135th base sequence of SEQ ID NO: 1 and the 156th to 156th sequence of SEQ ID NO: 2, and New Zealand deer deer antler. A first probe consisting of a nucleotide sequence of SEQ ID NO: 9 for discrimination, and a second probe consisting of a nucleotide sequence of SEQ ID NO: 10 for discriminating Chinese antler;
The second composition comprises a primer set and a Russian deer antler comprising a second primer pair for amplifying the 564 th base sequence from SEQ ID NO: 3 to the 581 th base sequence and the 681 th base sequence to the 700 th base sequence of SEQ ID NO: 4 A third probe comprising a nucleotide sequence of SEQ ID NO: 11 for discrimination, and a fourth probe consisting of SEQ ID NO: 12 for discriminating elk antler,
The first to fourth probes are labeled with a fluorophore at the 5 ′ end and a quencher at the 3 ′ end.
The method of claim 9, wherein the fluorescent material labeled at the 5 ′ end of the first to fourth probes is different for each probe.
The method of claim 9, wherein the analyzing of the fluorescence includes performing a fluorescence melting curve analysis while gradually raising the temperature.
Wherein said fluorescence fusion curve analysis comprises measuring a temperature at which the intensity of fluorescence rapidly decreases (Melting temperature).
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