KR101675980B1 - Primer set for determining identity of plant materials in food, method of determining identity of plant materials in foods using the same and kit comprising the same - Google Patents
Primer set for determining identity of plant materials in food, method of determining identity of plant materials in foods using the same and kit comprising the same Download PDFInfo
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- KR101675980B1 KR101675980B1 KR1020150063273A KR20150063273A KR101675980B1 KR 101675980 B1 KR101675980 B1 KR 101675980B1 KR 1020150063273 A KR1020150063273 A KR 1020150063273A KR 20150063273 A KR20150063273 A KR 20150063273A KR 101675980 B1 KR101675980 B1 KR 101675980B1
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Abstract
Description
The present invention relates to a primer set for discriminating authenticity of a plant raw material in food, a method for determining authenticity of a plant raw material in food using the same, and a kit comprising the primer set. More particularly, 2, a primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the primer pair of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6, and the primer pair of SEQ ID NO: A primer set for discriminating authenticity of vegetable raw materials in food, a method for determining authenticity of a plant raw material in food using the same, and a kit comprising the primer pair, wherein the primer pair comprises a forward primer and a reverse primer of SEQ ID NO: .
The Chinese medicinal herb medicine and medicinal herb contains the mushroom ( Cynanchum wilfordii hemsley), bovine ancestor (耳 葉 牛皮 消, Cynanchum auriculatum Royle ex Wight and Cynanchum bungei (9,13,14). In Korea, "Korean Pharmacopoeia (Herbal Medicine) Specification Collection" of Korea, only the origins of the origins of plants are prescribed.
In Korea, silver molluscs, widely cultivated in Korea, have not been used for farming because of low cost and labor, but also because of low productivity (13,14). In the early 1990s, , Most of the farms have been cultivated in this farm instead of silver mockery.
Hansuo, which is found in Korea, China, Japan, and North Korea, has Polygonum multiflorum as its origin and Cynanchum wilfordii as the origin plant in the Korean and North Korean pharmacopoeia or standard form. not.
There is a problem that the regulations and the reality do not coincide with each other, and research is urgently needed to solve them (3, 14). In addition, P. multiflorum Thunberg is a medicinal herb that is mixed in the name of Hashuo in the herbal medicine market, folk medicine, and clinic in Korea despite its different taxonomic position and effective ingredient.
As well as o white water (mock), and the right yiyeop accused are milkweed (Metaplexis japonica Makino) is very similar to the fruit and seed of the mature stage, which causes confusion among those interested in herbal medicine.
The tinnitus of Baeksoo was abused as Baekseoso, but it should not be confused with Baeksoo and Hassuo because their origin and content are completely different. It should be noted that since it is distributed in domestic market, it is mixed with white algae.
This leaflet is long cylindrical or spindle shaped, and its shape is slightly similar to that of Baekgoo (12,14,20), but it is a non-food raw material due to its origin and nonconformity. (FDA Poisonous Plant Database; http: //www.accessdata.fda . Gov / scripts / Plantox /Detail.CFM?ID=11513).
In addition, Cynanchum boudieri is often called a leafhopper , imported from China, and cultivated. As in the case of Yiwoo Woo, the Jiwang Wu appeal is not classified as Chinese medicine in Korea.
It is very difficult to distinguish whether or not it is mixed because it is processed in the form of powder or extract, because it is difficult to distinguish it from the general public because it uses a large root, which is used as a raw material of health food and supplements .
In order to accurately and quickly identify plant species, methods utilizing DNA markers that are not affected by external factors have recently been actively studied for major crops.
Unlike the morphological characteristics such as the shape and size of the plant, the method using the DNA marker can distinguish the plant species without being influenced by the external environment, and it is possible to discriminate the plant because there is no limit on the number of available markers (9-11 , 14).
The development of molecular biology and genetic analysis techniques have used species-specific PCR methods that can distinguish species within the same species, as well as species identification based on species-specific DNA sequence differences (15,21,22,23). Also, a method for identifying a herbicide-tolerant soybean plant (Korean Patent Laid-Open No. 10-2012-0107479), a primer set for a flowering-type plate of a Chinese cabbage plant and a discriminating method using the same (Korean Patent Publication No. 10-2012-0124945 ), Primers for PCR assay for transgenic plants (Korean Patent Laid-Open No. 10-2002-0092034), and the like.
On the other hand, genes are present in all the tissues of plants and plants, and the stability against pressure and high heat treated in the food manufacturing process is relatively higher than that of protein. Among the methods using genes, it is most common to amplify a specific gene region using a common primer and then to identify the correct species by base sequence analysis. However, the time for analyzing the nucleotide sequence is relatively long and the nucleotide sequence information Is not registered, it is difficult to identify the exact sample.
In addition, since relatively long template DNA is required, there is a limit to the discrimination of food ingredients in heat-treated processed foods that may be damaged by heat. Recently, interest in species discrimination using species-specific primers has been increasing, and primers that amplify in response to specific nucleotide sequences have been developed and used to judge the authenticity of plant raw materials in foods (1,16,18,19, 24,25).
However, these conventional techniques have limitations in determining whether or not the vegetable raw materials such as the sewage sludge, white sludge, white sludge, and saury wastes are true or false.
The present inventors have continuously conducted studies to solve the problems of the prior art as described above. For the identification of the species of Hashuo, Baishuo, Yibou, and Zhengwuo, Specific primers have been developed and the optimal PCR conditions have been established, it has been found that the authenticity of the vegetable raw materials in food can be judged, and the present invention has been completed.
Accordingly, an object of the present invention is to provide a primer set which can efficiently discriminate authenticity of a plant raw material in food.
It is another object of the present invention to provide a method for efficiently discriminating authenticity of a plant raw material in food using the primer set.
It is still another object of the present invention to provide a kit capable of efficiently discriminating authenticity of a plant raw material in food using the primer set.
In order to achieve the above object, the present invention provides a primer pair comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, a primer pair of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: A primer set for discriminating the authenticity of a plant material in food, comprising a pair of primers consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6, and a pair of primers consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: to provide.
In addition, the present invention provides a method for determining authenticity of a plant raw material in food using the primer set.
Further, the present invention provides a kit for discriminating authenticity of a plant raw material among foods containing the primer set.
According to the present invention, it is possible to confirm the authenticity of a raw material using a species-specific primer in a simple and accurate manner not only for a sample raw material but also for a processed food which is hard to be visually confirmed, .
FIG. 1A shows the PCR results of various samples by PCR using a haploid specie primer. In Fig. 1A,
FIG. 1B shows the PCR results of various samples by PCR using the specific primers. In Fig. 1B,
Fig. 1C shows PCR results of various samples by PCR using the bifidobacterial species specific primer. In Fig. 1C,
FIG. 1D shows the results of PCR of various samples by PCR using a specific primer of the genus Asiaticus. In Fig. 1 (d),
FIGS. 2A and 2B show the results of PCR confirmation experiment by mixing the Bacillus subtilis and Bacillus subtilis. FIG. 2A shows a specific primer of Bacillus subtilis, and FIG. 2B shows a specific primer of Bacillus subtilis.
FIG. 3 shows the results of comparison and analysis of the nucleotide sequence of a gene ( psbA - trnH ) for designing a pseudo- species specific primer.
FIG. 4 shows the results of comparison and analysis of the nucleotide sequence of the gene ( psbA - trnH ) for designing a primer specific primer.
FIG. 5 shows the results of comparison and analysis of the nucleotide sequence of the gene ( psbA - trnH ) for the bifid specific primer design.
FIG. 6 shows the results of comparison and analysis of the nucleotide sequence of the gene ( psbA - trnH ) for the design of the primers for interspecific primers.
The primer pair comprising the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, the primer pair of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4, the forward primer of SEQ ID NO: 5, And a pair of primers comprising a forward primer of SEQ. ID. NO. 7 and a reverse primer of SEQ. ID. NO. 8, and a primer set for discriminating authenticity of a plant raw material in food.
In the present invention, a "primer" is a single strand of oligonucleotides, which is hybridized under suitable conditions (presence of four different nucleoside triphosphates and a polymerase such as DNA or RNA polymerase), a template- Quot; means acting as a starting point at which to initiate directed DNA synthesis.
In the present invention, the suitable length of the primer is determined by the characteristics of the primer to be used, but is usually 15 to 30 bp. The primer need not be exactly complementary to the sequence of the template, but should be complementary enough to form a hybrid-complex with the template.
In the primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 is a polygonum multiflorum ).
In the primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 is Cynanchum wilfordii ).
In the primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 may be used for discrimination of Cynanchum . Auriculatum .
In the above primer set of the present invention, the primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 is Cynanchum boudieri ).
In the primer set of the present invention, a primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 generates a PCR amplification product of 160 bp in size.
In the primer set of the present invention, a primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4 generates a 147-bp PCR amplification product.
In the primer set of the present invention, a primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 generates a 119-bp PCR amplification product.
In the primer set of the present invention, a primer pair consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 generates a PCR amplification product of 127 bp in size.
In the primer set of the present invention, the plant may be at least one member selected from the group consisting of Hashuo, Bacillus subtilis, Bacillus subtilis, and Bacillus subtilis.
The present invention also relates to a method for determining authenticity of a plant raw material in food using the primer set.
A method for determining authenticity of a plant raw material among the foods of the present invention comprises the steps of (a) extracting template DNA from a sample, (b) amplifying the template DNA extracted in the above step with a polymerase chain reaction And (c) fractionating the amplification product produced by the step, thereby confirming authenticity of the vegetable ingredient in the food.
In the method of the present invention, the plant may be at least one selected from the group consisting of Hashuo, Bassuo, bamboo, and bamboo.
Hereinafter, the method of the present invention will be described in more detail.
(a). From the sample Genomic template DNA ( genomic DNA ),
The separation of genomic DNA from a food sample in the method of the present invention can be carried out according to a conventional method known in the art, for example, a Phenol-Chloroform extraction method (Miller et SA, Dykes DD, Polesky HF Nucleic Acids Res. 16, p 1215, 1998).
(b). remind primer The molds extracted in the above step DNA To polymerization Amplification by enzyme chain reaction
Using the genomic DNA prepared in the step (a) as a template, PCR is performed using the primer set of the present invention.
The polymerase chain reaction is a method for selectively amplifying a specific DNA fragment, for details, see Miller, H. I. (WO 89/06700) and Davey, C. et al. (EP 329,822), the disclosures of which are incorporated herein by reference. In the polymerase chain reaction, various DNA polymerases can be used for the amplification reaction, including, for example, the "Klenow fragment" of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase do.
Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) .
In the present invention, the amplification reaction solution may include a dNTP mixture (dATP, dCTP, dGTP, dTTP), a PCR buffer, and a DNA polymerase joiner.
On the other hand, when the amplification reaction by the polymerase chain reaction is carried out, it is preferable to provide the reaction container with an excessive amount of components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component.
It is required to provide the reaction mixture with such a partner as Mg 2 + , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions.
Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.
In the present invention, the amplification reaction with a polymerase can be carried out by a cycle consisting of (i) a pre-denaturation process, (ii) annealing, elongation and denaturation several times to several tens of times (Iii) a final heat treatment process or a thermal cycle program suitably modified for these processes.
A primer pair consisting of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 generates a PCR amplification product of 160 bp in size.
In the primer set, a PCR amplification product of 147 bp in size is generated by the primer pair consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4.
A primer pair consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6 generates a PCR amplification product of 119 bp in size.
Also, a pair of primers consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8 generates a PCR amplification product of 127 bp in size.
(c). The < RTI ID = 0.0 > The amplified product By fractionation In food Step of discriminating authenticity of vegetable raw material
The PCR amplified products of the four primer sets of the present invention all form DNA amplification product bands of different sizes. PCR amplified DNA amplification products can be observed by methods known in the art, for example, electrophoresis on agarose gel and EtBr staining and visualization by ultraviolet irradiation.
The present invention also provides a kit for discriminating authenticity of a plant raw material among foods containing the primer set.
When the kit for determining the authenticity of a plant raw material among the foods of the present invention is applied to a PCR amplification process, a reagent necessary for PCR amplification such as a buffer, a DNA polymerase (for example, Thermus aquaticus (Taq), Thermus thermophilus ), Thermus filiformis, Thermisflavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs.
The kit for discriminating authenticity of vegetable raw materials among the foods of the present invention may be manufactured from a number of separate packaging or compartments containing the above reagent components.
In the kit for discriminating the authenticity of a plant raw material among the foods of the present invention, the plant may be at least one selected from the group consisting of Hashuo, Baishuo, bamboo, and bamboo.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.
<Examples>
1. Materials and Methods
(One). Sample preparation and pretreatment
The sewage (Polygonummultiflorum), beasts O (Cynanchumwilfordii), yiyeop right sues (C. auriculatum) and section Guan accused (Cynanchum used in this embodiment boudieri ) were used as a standard sample in the standard drug preparation and the standardized herbal medicine collected from the Graduate School of Medicinal Products for Safety Evaluation of the Food and Drug Administration . Four kinds of Sasao (four kinds), white water (five kinds) Were randomly selected by product type and purchased.
In order to confirm the result of mixing with standard samples, the mixture was mixed with 0%, 0.5%, 1%, 2%, 5%, 10% and 50% (w / w) Each gene was extracted and the juice form of the product was centrifuged at 20,000 xg for 5 minutes to remove the supernatant, and only the precipitate was dried and used as a sample.
(2). Gene extraction
The DNeasy Plant mini kit (Qiagen GmbH, Hilden, Germany) was used for the gene extraction and the extraction method was extracted according to the method provided by the manufacturer.
(3). Gene simple amplification
For gene amplification, whole genome amplification kit (WGA, SIGMA, USA) was used to amplify a small amount of gene extracted by heating and pressing under product processing during primer application.
Amplification was performed according to the method provided by the manufacturer. The final amplified products were purified using AccuPrep PCR Purification Kit (Bioneer, Korea) and used in PCR experiments.
(4). Species specific primer design
For the design of species specific primers, the chloroplasts and nuclear genetic information on the Hsuo, Baishuo, Yibeo, and Zhenggui faeces registered in the Gene Bank operated by the National Institute of Health (NOW) were confirmed.
In the absence of genetic information, the gene was amplified using a common primer, and the nucleotide sequence was determined to design a species specific primer, which was synthesized by Bioneer, Korea.
(5). PCR reaction and confirmation of results
Composition of the reaction solution for PCR was extracted
For gene amplification, Taq. DNA polymerase, dNTPs and MgCl 2 were purchased from TAKARA (TaKaRa Co., JAPAN), and the PCR instrument was a C1000 Touch ™ Thermal Cycler (Bio-RAD Laboratories, Inc. USA).
To confirm the final product, 5 μl of the reaction solution was subjected to electrophoresis at 100 V for 30 minutes with agarose gel (1 μl / ml) to which ethidium bromide (EtBr) was added. The size of the PCR product was determined using a 100 bp DNA ladder (Bioneer, Korea) and the results were confirmed using a UV projector after completion of the electrophoresis.
2. Results and discussion
(One). Species specific primer design
In this embodiment, a species specific primer capable of discriminating authenticity of Hashuo, Baishuo, Yibeo, and Zhenguo was developed.
For the species specific primer design, the chloroplast and nucleotide genetic information for the Hsuo, Baishuo, Yibeo, and Zhenggui faeces registered in the Gene Bank operated by the National Institute of Health were confirmed. In the absence of genetic information, (Universal primer) was used to amplify the gene and sequenced, and then a species specific primer was designed.
Among the genes used as DNA barcoding markers in the species identification studies, the
Since the product is processed and processed into processed food such as health functional foods, the specially designed primer is designed to have a PCR product size of about 200 bp considering the application to processed foods. The results are shown in the following table Respectively.
designation
(bp)
gene
psbA
-
trnH
(2). Optimize and verify PCR conditions
PCR was performed under optimized conditions using each species-specific primer, and the results are shown in Table 2 below.
Polygonummultiflorum , Cynanchumwilfordii , C. auriculatum and Cynanchum boudieri were able to identify amplified products of expected sizes of 160, 147, 119 and 127 bp, respectively, Non-specific bands were not produced in the comparative species other than the target species of the present invention (see Figs. 1A to 1D).
FIG. 1A shows the PCR results of various samples by PCR using a haploid specie primer. In Fig. 1A,
FIG. 1B shows the PCR results of various samples by PCR using the specific primers. In Fig. 1B,
Fig. 1C shows PCR results of various samples by PCR using the bifidobacterial species specific primer. In Fig. 1C,
FIG. 1D shows the results of PCR of various samples by PCR using a specific primer of the genus Asiaticus. In Fig. 1 (d),
In addition, in a mixed confirmation experiment of Bacillus subtilis and Bacillus anthracis, when the bacillus annuae of 2% or more was mixed, the bacillus gene was confirmed by PCR (see FIG. 2).
FIGS. 2A and 2B show the results of PCR confirmation experiment by mixing the Bacillus subtilis and Bacillus subtilis. FIG. 2A shows a specific primer of Bacillus subtilis, and FIG. 2B shows a specific primer of Bacillus subtilis.
In Figs. 2A and 2B,
FIG. 3 shows the result of comparison and analysis of the nucleotide sequence ( psbA - trnH ) for the design of the specific primer of the dung species. FIG. 4 shows the nucleotide sequence of the gene ( psbA - trnH ) The results are as follows.
5 shows the result of comparison and analysis of the nucleotide sequence ( psbA - trnH ) for the primer design of bifidobacterial species specific primer. Fig. 6 shows the results of the comparison of gene ( psbA - trnH ) base Sequence comparison and analysis.
(3). Application to processed foods
In order to confirm the applicability of the species specific discrimination method for Husuo, Baeksoo, Yiwabu, Woojisu, and Zenyuu, we used 9 randomly selected processed foods (4 Hashuo products, 5 Baishuo products) And then analyzed.
Due to the high temperature and pressure of the product, small amounts of genes were extracted and the amount of DNA extracted by product type (juice, powder) was different. Therefore, the gene was simply amplified using the WGA kit and used in the experiment. The results are shown in Table 3 below.
As shown in Table 3, in the case of Sasao, the gene of Sasao was confirmed in the juice form and the powder form, and the Baishuo gene was also confirmed in the two products. In addition, all five genes were identified in the product.
On the other hand, the genes for the bovine endosperm and the bovine endosperm were not identified in the products of Hashuo and Hissho.
As can be seen from the above, the species specific primers developed through the present embodiment showed improved species discrimination methods for raw materials and processed products of Hashuo, Baishuo, Yiwu, The cause of the cross-validation cases shown could not distinguish between unintentional inclusion due to the same production line or intentional mixture to reduce unit costs.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the present invention is not limited to the disclosed exemplary embodiments, but many variations and modifications can be made by those skilled in the art within the technical scope of the present invention. Will be self-evident.
According to the present invention, it is possible to confirm the authenticity of a raw material using a species-specific primer in a simple and accurate manner not only for a sample raw material but also for a processed food which is hard to be visually confirmed, Therefore, the present invention can be applied to a technical field to which the present invention belongs.
<110> Republic of Korea (Ministry of Food and Drug Safety) <120> Primer set for determining identity of plant materials in food, method of determining identity of plant materials in foods using the same and kit comprising the same <130> 10093 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer of PM-F <400> 1 aagttttcct taccttaccc atta 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of PM-R <400> 2 atattatatt ctaaaattag at 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer of CW-F <400> 3 atattatatt ctaaaattag at 22 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of CW-R <400> 4 ctctatttct atttctat 18 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer of CA-F <400> 5 aattaattt aaaaattcaa taca 24 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of CA-R <400> 6 gttctatttc tatttatttt tat 23 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer of CB-F <400> 7 tatttttata taaaaattat aaaatatta 29 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of CB-R <400> 8 cctattgtaa tttattaatt tt 22
Claims (11)
Wherein the vegetable raw material is at least one selected from the group consisting of Hashuo, Bacillus, Bacillus subtilis, and Bacillus subtilis; and a primer set for judging whether the plant material is genuine or not.
Wherein the vegetable raw material is at least one selected from the group consisting of Hashuo, Baishuo, bamboo, and bamboo, and determining the authenticity of the vegetable raw material in the food.
(a) extracting genomic DNA from a sample;
(b) amplifying the template DNA extracted in the above step using the primer set by a polymerase chain reaction; And
(c) fractionating the amplification product produced by the above step to confirm whether or not the plant raw material is authentic in the food.
Wherein the vegetable raw material is at least one selected from the group consisting of Hashuo, Baishuo, bamboo, and bamboo.
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| KR20210081498A (en) * | 2019-12-23 | 2021-07-02 | 대한민국 (식품의약품안전처장) | Composition for distinguishing a plant of genus Cynanchum |
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| KR20090123113A (en) * | 2008-05-27 | 2009-12-02 | 한국 한의학 연구원 | Dna marker for discrimination of polygonum multiflorum thunberg, cynanchum wilfordii max. hemsl. and cynacum auriculatum royle ex wight |
| KR20120062614A (en) * | 2010-12-06 | 2012-06-14 | 재단법인 제주테크노파크 | Method for discriminating between cynanchum wilfordii and cynanchum auriculatum and a kit therefor |
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| KR102284051B1 (en) * | 2019-12-23 | 2021-08-02 | 대한민국 | Composition for distinguishing a plant of genus Cynanchum |
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