WO2006016762A1

WO2006016762A1 - A method for genetic identification of ginseng species and the kit using thereby

Info

Publication number: WO2006016762A1
Application number: PCT/KR2005/002572
Authority: WO
Inventors: Jung Chul Seo; Kang Hyun Leem; Joo Ho Chung; Dong Hoon Shin; Kang Duk Choi; Chae Ha Yang; Sang Chan Kim; Sang Won Han; Byung Cheol Lee; Sung Ki Jung
Original assignee: Bio-Medic Research Center Daegu Hanny University
Priority date: 2004-08-13
Filing date: 2005-08-08
Publication date: 2006-02-16

Abstract

The present invention relates to a method for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method using by species-specific sequencing primer for ginseng designed by the present inventors, and the kit using thereby which can identify the species of ginsengs concretely and rapidly. Therefore, those method and the kit using by the species-specific sequencing primer for ginseng can be useful in discriminating required ginseng species, providing accurate ginseng material, controlling the quality of ginseng products and used as fundamental material to discriminate other natural resources.

Description

A METHOD FOR GENETIC IDENTIFICATION OF GINSENG SPECIES AND THE KIT USING THEREBY

Technical Field

[1] The present invention relates to a method for identifying ginseng species and the kit using the method. More particularly, the present invention relates to a method for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method and the kit using thereby.

[2]

Background Art

[3] It is known that there are many Panax species belonged to Araliaceae, for example,

Panax ginseng distributed or cultivated in far-eastern Asia region, for example, Panax quinquefolia in America and Canada, Panax notoginseng in China, Panax trifolia in eastern region of north America, Panax japonica in Japan, China and Nepal, Panax pseudoginseng in Nepal, Panax vietnamensis in Vietnam, Panax elegatior, Panax wangianus and Panax bipinratifidus etc. The ginseng root has been used in traditional Korean and Chinese medicine for a long time (Kim, H.C., "Herbal Pharmacology" 1st Ed., Jibmoon Press, Seoul, pp. 422-427, 2001). The herbal supplement market has been growing rapidly in recent decades. Panaxis one of the most medicinally important genera in Oriental medicine. It is one of the approximately 120 genera of plants with an eastern Asian and eastern North American disjunct distribution (Wen, J., and Zimmer, E. A., Phylogeny and biogeography of Panax L. (the ginseng genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. MoI. Phylogenet. Evol., 6, ppl67-177, 1996). Two of the species, i.e., Panax ginseng, known as Korean ginseng, and Panax quinquefolius, American ginseng are regarded as important medicines in Korea and China (Bhattaram, V.A. et al, Pharmacokinetics and bioavailability of herbal medicinal products. Phytomedicine, 9 (Suppl 3), ppl-33, 2002). Of the two species, Panax ginseng is thought to tonify the Qi (energy )more ef¬ fectively against aging, weakness, and stress than Panax quinquefolius. In the Korean market, the price of Panax ginseng is much higher than that of Panax quinquefolius. A few dealers have practiced the deception of disguising Panax quinquefolius as Panax ginseng, because the general consumer could not distinguish between the two whereas other ginseng species, including Panax notoginseng and Panax japonica, can be easily identified. There have been several differences in pharmacological potency, cost etc between Panax quinquefolius as Panax ginseng, i.e., Panax ginseng has more potent medicinal effects in respect to anti-aging, anti-fatigue anti-stress etc than that of Panax quinquefolius although the former is relatively higher cost comparing with the latter. However, many commercial ginseng products are extremely difficult to identify in the form of slice, powder or extract. Identification via analysis of chemical profiles is also very difficult due to many variables, such as the soil condition, climate, and nutritional factors.

[4]

[5] Therefore, many merchandisers have been used to cheating in selling ginseng products for obtaining economical interest and there have been difficulty in de¬ termining or discriminating true ginseng product among other false ginseng products in the form of powder or slice form (Um J. Y. et al, Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP. Biol. Pharm. Bull, 24(8), pp872-5, 2001). Furthermore, the discrimination of ginseng species using by chemical analysis is also difficult because of its variety, i.e., earth condition, weather, nutritional factor etc (Mihalov JJ. et al, DNA identification of commercial ginseng samples. J. Agric. Food Chem., 48(8), pp3744-52, 2000). Gel-based method, a conventional ginseng dis¬ criminating method using by genetic analysis, also has several problems such as a possibility of false result according to examiner's false subjective judgment.

[6] The internal transcribed spacer region (ITS) of nuclear ribosomal DNA is known to be a good molecular site for analysis of species in plants (Baldwin, B. G., et al, The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann. Mo. Bot. Gard, 82, 247 277, 1995); White, T. J., et al, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications, eds. Innis, M., Gelfand, D., Sninsky, J., and White, T., Academic Press, San Diego, pp. 315 322, 1990), we studied the genetic variance between the two species using pyrosequencing analysis of ITS (Wieser, F. et al; Analysis of an interleukin-6 gene promoter polymorphism in women with endometriosis by pyrosequencing. J. Soc. Gynecol. Investig., 10, pp32-36 2003).

[7] However, there have been no disclosure or suggestion on a method for identifying the ginseng species by discriminating specific gene of ginseng according to py¬ rosequencing method till now.

[8] Present inventors have been studied to determine whether two ginseng species (

Panax ginseng and Panax quinquefolius) can be identified by genetic analysis or not and to verify pyrosequencing analysis used to assess genetic variation. The py¬ rosequencing results constituted clear data showing different pattern between each other. Accordingly, we have found that pyrosequencing analysis can be able to identify the Panax species and finally have completed the present invention by confirming the mutation of Single Nucleotide Polymorphism (SNP) in the DNA extracted from Panax species using by pyrosequencing method, a recently developed DNA sequencing tech nique used in evaluating genetic mutation. [9]

Disclosure of Invention Technical Problem

[10] Accordingly, it is an object of the present invention to provide a method for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method.

[11] It is another object of the present invention to provide a kit for identifying the ginseng species using by pyrosequencing method.

[12]

Technical Solution

[13] The present invention provides a method for identifying the ginseng species by dis¬ criminating specific gene of ginseng according to pyrosequencing method.

[14] The present invention provides a method for identifying the ginseng species by dis¬ criminating specific genes of ginseng according to pyrosequencing method comprising the steps consisting of: extracting DNA from ginseng sample at 1^st step; preparing reaction solution comprising the extracted DNA and primer at 2^nd step; amplifying the reaction solution at 3 step; analyzing total base sequence of the amplified DNA at 4 step; and identifying ginseng species through genotyping analysis obtained by subjecting to pyrosequencing method using by combination with the amplified DNA.

[15] The method of the present invention can provide rapid discrimination using by small quantity of sample and provide more exact diagnosis without subjective factor than conventional gel based method as a non-gel based method.

[16] The present invention provides a kit for identifying the ginseng species by dis¬ criminating specific gene of ginseng according to pyrosequencing method.

[17] Specifically, the present invention provides a kit for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method comprising the steps consisting of: extracting DNA from ginseng sample at 1^st step; preparing reaction solution comprising the extracted DNA and primer at 2^nd step; amplifying the reaction solution at 3^rd step; analyzing total base sequence of the amplified DNA at 4 step; and identifying ginseng species through genotyping analysis obtained by subjecting to pyrosequencing method using by combination with the amplified DNA.

[18]

[19] More specifically, above described ginseng samples at 1^st step comprise all the parts such as root, stem, and leaf of any of Panax species.

[20] At 2ⁿ step in the above-described method, biotinylated primer comprising the primer represented by SEQ ID: 1 may be preferably used as a sense primer and the general primer comprising the primer represented by SEQ ID: 2 may be preferably used as an antisense primer for DNA amplification. The biotinylated primer defined herein could be designed to have 18 to 20 mer.

[21] At 5 step in the above described method, the primer used in pyrosequencing method comprises specific sequencing primers to the species of ginseng sample represented by SEQ ID: 3 for genotype analysis, wherein the primer is designed to have 10 to 15 mer in order to the primers should not to form the secondary structure and to be present in the adjacent sites to mutation sites.

[22] In the pyrosequencing method at 5th step, specific light emits where nucleotide is synthesized from extended DNA fragment and the genotype of ginseng polymorphism is automatically identified. The method is subjected by microtiter-based pyrosequencer instrument based on automatic micro-titer which can provide sample analysis with rapid and quick completion within about 15 mins and provide respective nucleotide synthesis with rapid completion within about 1 min. Accordingly, the method could be used in determining the exact sequence of genetic mutation in rapid.

[23] The present invention provides a kit comprising a sense primer represented by SEQ

ID: 1, anti-sense primer represented by SEQ ID: 2 and a specific sequencing primer to the species of ginseng sample represented by SEQ ID: 3 for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method.

[24] The present invention also provides a kit for identifying the ginseng species by dis¬ criminating specific gene of ginseng characterized in using a standard analysis drawing data and the result table as shown in Fig. 1 and Fig. 2 resulting from above-described pyrosequencing method.

[25] The method and kit of the present invention can discriminate the ginseng genus among other ginseng species, for example, Panax ginseng distributed or cultivated in far-eastern Asia region, Panax quinquefolia in America and Canada, Panax no- toginseng in China, Panax trifolia in eastern region of north America, Panax japonica in Japan, China and Nepal, Panax pseudoginseng in Nepal, Panax vietnamensis in Vietnam, Panax elegatior, Panax wangianus and Panax bipinratifidus etc, preferably, Panax ginseng and Panax quinquefolia using by species-specific primer.

[26] The inventive method and kit of the present invention can provide with prompt dis¬ crimination using by small quantity of ginseng sample and more concrete diagnosis excluding subjective element as a non-gel based method than conventional gel based method.

[27]

[28] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[29]

Advantageous Effects

[30] The present invention can provide a method for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method using by species-specific sequencing primer for ginseng designed by the present inventors, and the kit using thereby which can identify the species of ginsengs concretely and rapidly. Therefore, those method and the kit using by the species-specific sequencing primer for ginseng can be useful in discriminating required ginseng species, providing accurate ginseng material, controlling the quality of ginseng products and used as fundamental material to discriminate other natural resources.

[31]

Brief Description of the Drawings

[32] Fig. 1 shows the result of pyrosequencing method for Panax ginseng;

[33] Fig. 2 shows the result of pyrosequencing method for Panax quinquefolius.

[34]

Best Mode for Carrying Out the Invention

[35] It will be apparent to those skilled in the art that various modification and variation can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[36] The present invention is more specifically explained by the following figures and examples. However, it should be understood that the present invention is not limited to these examples in any manner.

[37]

Mode for the Invention

[38] The following Reference Example, Example and Experimental Examples are intended to further illustrated the present invention without limiting its scope.

[39]

[40] Reference Example 1. Preparation of ginseng sample

[41]

[42] Two different species of ginsengs, i.e., Korean ginseng (Panax ginseng) sample purchased from Cheon-buk Jinan Nong-hyup loctated at Jinan city in Korea and American ginseng (Panax quinquefolia) cultivated at Liaoning region in China and au- thentified by the professor majored in herbology, were prepared.

[43]

[44] Example 1. DNA Purification of ginseng sample [45]

[46] 3 g of ginseng sample comprising whole root part was minced to slices with sterilized cutter, crushed with sterilized crusher and pulverized. Modified DNA isolation kit (DNeasy No. 69104, Qiagen Inc., Valencia, CA, USA) was used according to the manuals of manufacturer. 300 D of pulverized ginseng samples were washed with several times. Prior to eluting step of samples, column was dried at 37 °C for 5 mins to evaporate remaining ethanol and then the samples were eluted with 200 D of total volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

[47]

[48] Experimental Example 1: DNA amplification using by Polymerase Chain

Reaction (PCR) method

[49]

[50] The amplification of extracted DNA was performed by PCR method as follows. ITS

(Internal Transcribed Space) regions was amplified by using 25 ng of DNA and 5 pmol of each primer (forward: SEQ ID: 1, Reverse: SEQ ID: 2). The amplification of PCR was performed by using 0.5 unit Taq polymerase (HT Biotechnology Ltd., Cambridge, United Kingdom). 30 D of PCR reaction mixture was prepared by mixing 10 mM Tris- HCl (pH 9.0), 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.25 mM of each 0.1 % Triton-X 100, 0.01 % (v/v) stabilizer, dNTP (deoxynucleotide triphosphate) and 0.1 M oligonucleotide primer together. PCR reaction was performed by denaturing the DNA at 95 °C for 5 mins and subjecting repeated heating steps i.e., heating at 95 °C for 30 seconds as the 1^st step, 60 °C for 30 seconds as the 2ⁿ step and 72 °C for 30 seconds as the 3^rd step, at 30 times. The biotinylated primer combinable with single strand DNA was used as a reverse primer. The confirmation of PCR product was determined by 1.5 % agarose gel electrophoresis.

[51]

[52]

[53] Experimental Example 2: Genotyping using by pyrosequencing method

[54] DNA preparation using by pyrosequencing method was performed in accordance to the standard protocol of manufacturer (Pyrosequencing AB, Uppsala, Sweden). Streptavidin sepharose beads (Streptavidin sepharose HP, Amersham Pharmacia Biotech, Uppsala, Sweden) were fixed to PCR product. The sequencing primer of ginseng sample was SEQ ID: 3 prepared by hybridization with terminal residue adjacent to the proximal end of T/C mutation using by pyrosequencing AN ( http://www.pyrosequencing.com^') as shown in Table 1. The primer was left alone at room temperature for 10 mins. 20 D of biotinylated PCR product was fixed to streptavidin-coated sepharose and the fixed PCR product was transferred to 96-well filter (Millipore, Bedford, MA, USA). The beads were remained in the wells and remaining other solution and reagent were removed. Pure single stranded DNA was obtained by vacuum condition. Template fixed beads were dipped into 55 D of 4 M acetic acid containing 0.35 μM sequencing primer and 45 D of floaters were transferred to PSQ 96 plates (Pyrosequencing AB, Uppsala, Sweden). The PSQ 96 plates containing sample were heated at 90 °C for 5 mins to anneal the sequencing primer using by PSQ 96 sample prep thermoplate, left alone for 10 mins at room temperature and transferred to the process chamber of PSQ 96 instrument (Pyrosequencing AB, Uppsla, Sweden). Each enzyme, substrate and nucleotide was produced in each well from the reagent cassette using by PSQ 96 SNP reagent kit (Pyrosequencing AB, Uppsala, Sweden). The light emitted where the nucleotide was synthesized from extending DNA strands. The genotype of ginseng polymorphism was confirmed by the above-described procedure.

[55] [56] Table 1

[57] [58] To determine whether two ginseng species can be identified by pyrosequencing method, we performed pyrosequencing analyses of Panax ginseng cultivated in Korea and of Panax quinquefolius cultivated in China or U.S. A. The pyrosequencing results constituted clear data. Panax quinquefolius showed a very different pattern than Panax ginseng. The peak of Panax quinquefolius was absent in the first T nucleotide base and the peak of the next C nucleotide base was higher than that of the next G nucleotide base (Fig. 1). But in Panax ginseng the peak of the first T nucleotide base was same height as that of the next C nucleotide base (Fig. 2). We designed ginseng species- specific sequencing primer to identify Panax ginseng and Panax quinquefolius. In the sequence, Panax ginseng has a T nucleotide base, but Panax quinquefolius has a C nucleotide base. Through above-describe results, we have verified that the ginseng species-specific sequencing primer of the present invention was well-designed.

[59] At present, pyrosequencing is performed in an automated microtiter-based py- rosequencer instrument, which allows simultaneous analysis of samples within 15 min. Each round of nucleotide dispensing takes approximately 1 min and thus offers a more rapid and convenient way to determine the exact sequence of the genetic variations than do other methods such as randomly amplified polymorphic DNA (RAPD), re¬ striction fragment length polymorphism (RFLP) etc.

[60]

[61] Accordingly, it is confirmed that the species-specific sequencing primer for ginseng was appropriately designed by the present inventors.

[62] The above-described method for identifying the species of ginseng and the species- specific sequencing primer for ginseng designed by the present inventors can identify the species of ginsengs concretely and rapidly. Therefore, those method using by the species-specific sequencing primer for ginseng can be useful in identifying required ginseng species, providing accurate ginseng material, controlling the quality of ginseng products and used as fundamental material to discriminating other natural resources.

[63]

[64] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

[65]

Industrial Applicability

[66] As described above, the method and the kit using thereby for identifying the species of ginseng and the species-specific sequencing primer for ginseng designed by the present inventors can identify the species of ginsengs concretely and rapidly. Therefore, those method and the kit using by the species-specific sequencing primer for ginseng can be useful in identifying required ginseng species, providing accurate ginseng material, controlling the quality of ginseng products and used as fundamental material to discriminating other natural resources.

[67]

Sequence Listing

[68] SEQ ID 1 : 5 ' -CC A AGGAAATC AAACTGAAC-3 ' is sense primer of ginseng sample, SEQ ID 2: 5'-TCTGCAATTCACACCAAGTA-S' is anti-sense primer of ginseng sample and SEQ ID 3: 5'-GCCGAGATATCCGTT-3'is sequencing primer of ginseng sample.

Claims

[I] A method for identifying the ginseng species by discriminating specific genes of ginseng according to pyrosequencing method.

[2] A method for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method comprising the steps consisting of: extracting DNA from ginseng sample at 1^st step; preparing reaction solution comprising the extracted DNA and primer at 2" step; amplifying the reaction solution at 3^rd step; analyzing total base sequence of the amplified DNA at 4^th step; and identifying ginseng species through genotyping analysis obtained by subjecting to pyrosequencing method using by combination with the amplified DNA at 5^th step.

[3] The method according to claim 2 wherein said method is performed by SEQ ID:

1 or SEQ ID: 2 primer.

[4] The method according to claim 3 wherein said SEQ ID: 1 primer is biotinylated sense primer and SEQ ID:2 primer is anti-sense primer.

[5] The method according to claim 2 wherein said pyrosequencng method is performed by SEQ ID: 3 primer as a primer.

[6] The method according to claim 1 wherein said ginseng is selected from Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax trifolia, Panax japonica , Panax pseudoginseng, Panax vietnamensis, Panax elegatior, Panax wangianus or Panax bipinratifidus.

[7] The method according to claim 6 wherein said ginseng is Panax ginseng or

Panax quinquefolia.

[8] A kit for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method.

[9] The kit according to claim 8 wherein said kit comprises a sense primer represented by SEQ ID: 1, anti-sense primer represented by SEQ ID: 2 and a specific sequencing primer to the species of ginseng sample represented by SEQ ID: 3 for identifying the ginseng species by discriminating specific gene of ginseng according to pyrosequencing method.

[10] A kit for identifying the ginseng species by discriminating specific gene of ginseng characterized in using a standard analysis drawing data and the result table as shown in Fig. 1 and Fig. 2 resulting from pyrosequencing method.

[II] The kit according to claim 10 wherein said ginseng is selected from Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax trifolia, Panax japonica , Panax pseudoginseng, Panax vietnamensis, Panax elegatior, Panax wangianus and Panax bipinratifidus. [12] The kit according to claim 11 wherein said ginseng is Panax ginseng or Panax quinquefolia.