View
10
Download
0
Category
Preview:
Citation preview
J. Cent. South Univ. (2018) 25: 1052−1062 DOI: https://doi.org/10.1007/s11771-018-3805-9
Panax ginseng-specific sequence characterized amplified region (SCAR) marker for testing medicinal products
JIANG Qiu-tao(蒋秋桃)1, 2, LIU Li(刘丽)2, XIAO Bing-yi(肖炳燚)2, LI Wen-li(李文莉)2, LUO Hui-ming(罗晖明)2, NIE Ping(聂平)2, DING Ye(丁野)2, LI Jie(李洁)1, LI Wen-zhang(李文章)1
1. School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China;
2. Hunan Institute for Drug Control, Changsha 410001, China
© Central South University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract: To screen genetic polymorphisms of Panax ginseng, as well as those of Panax quinquefolium and Panax notoginseng, analysis of random amplified polymorphic DNA (RAPD) was performed using 120 random primers. Of the successful amplicons obtained, the Panax ginseng-specific RAPD marker C-12 was cloned into a TA vector and sequenced (GenBank access number KU553472). Based on the sequence analysis results, a pair of primers specific to C-12 was designed. Finally, a SCAR marker-based identification system for Panax ginseng was developed after optimization of the reaction conditions. Using this method, two positive bands were stably observed at 300 bp and 130 bp in 33 batches of Panax ginseng samples tested, while negative results were obtained for another 101 batches of samples, including Panax quinquefolium, Panax notoginseng, adulterants, and other medicinal herbs. Thus, we successfully developed a PCR-based method for rapid and effective identification of Panax ginseng, which can be effectively used for the protection and utilization of germplasm resources and identification of the origins of Panax ginseng samples. Key words: Panax ginseng; random amplified polymorphic DNA (RAPD); sequence characterized amplified regions (SCAR); molecular identification Cite this article as: JIANG Qiu-tao, LIU Li, XIAO Bing-yi, LI Wen-li, LUO Hui-ming, NIE Ping, DING Ye, LI Jie, LI Wen-zhang. Panax ginseng-specific sequence characterized amplified region (SCAR) marker for testing medicinal products [J]. Journal of Central South University, 2018, 25(5): 1052–1062. DOI: https://doi.org/10.1007/s11771-018- 3805-9.
1 Introduction
Ginseng drugs, including Ginseng (Panax ginseng C. A. Mey.), American ginseng (P. quinquefolium L.) and Notoginseng (P. notoginseng (Burk) F. H. Chen ex C. Chow & W. G. Huang) etc. are a group of valuable herbal medicines, which are widely used in Northern American and Asian countries, and have adaptogenic, restorative,
immunomodulatory, vasodilatory, anti-inflammatory, antioxidant, anti-aging, anticancer, anti-fatigue, anti-stress, and anti-depressive effects in rodents and humans [1–6]. Driven by economic development and the on-going internationalization of traditional Chinese medicinal herbs, ginseng drugs have been utilized not only in traditional treatment but also in health-food products and foods. Since ginseng is obtained from the same part of different plant species belonging to the same genus
Foundation item: Project(2014ZX09304307-002) supported by the Major Program of Science and Technology Foundation of China;
Project supported by Technology Platform for Quality/Safety Inspection and Risk Management of Traditional Chinese Medicine, China; Project(2014SK2001) supported by the Key Program Foundation of Hunan Provincial Science & Technology Department, China; Project(XSYK-R201502) supported by the Hunan Provincial Food and Drug Administration under Key Project of Science and Technology for Food and Drug Safety, China
Received date: 2017−04−26; Accepted date: 2017−10−30 Corresponding author: LI Wen-li, Professor; Tel: +86–731–82275866; E-mail: 1838675867@qq.com; LI Wen-zhang, Professor; Tel:
+86–13874992656; E-mail: liwenzhang@csu.edu.cn; ORCID: 0000-0002-1020-0554
J. Cent. South Univ. (2018) 25: 1052–1062
1053
and family, the above-mentioned three herbal medicines show highly similar internal structures and chemical compositions, making it very difficult to authenticate ginseng, particularly in single-drug or compound medicines. Unlawful practices such as substituting ginseng with other medicinal roots (e.g., the roots of Phytolacca acinosa Roxb. or Talinum paniculatum (Jacq.) Gaertn.) have become common in the market [7]. Counterfeit products do not have the claimed therapeutic effects and might even have toxic or adverse effects. The rapid adoption of DNA molecular marker-based technologies [8] to identify traditional Chinese herbal medicines and their decoction products has played an important role in authenticating medicinal products [9, 10].
Sequence characterized amplified region (SCAR) analysis [11], in which a single, genetically defined locus is identified by amplification of genomic DNA with a pair of specific oligonucleotide primers, is a highly sensitive and reliable molecular marker in various fields such as species identification, marker-assisted screening, and map-based gene cloning. In particular, SCAR is suitable for detecting differences in DNA among different samples. SCAR markers have the potential to replace RAPD (random amplified polymorphic DNA) and other DNA-based markers that are expensive, time-consuming, and tedious. SCAR tagging involves the recovery, cloning, and sequencing of specific RAPD fragments. This is followed by primer design based on the sequencing results, and the resulting primers are used to amplify genomic DNA by PCR. This method offers several benefits, including simplicity of operation, stable performance, and low cost [9, 12]. SCAR markers have been developed for authentication of various medicinal plants that can be easily adulterated. Currently, SCAR is used as a national standard for identification of pilose antler in Korean herbal pharmacopeia. ZHOU et al [13] obtained a SCAR marker specific for Polygonum capitatum (P. capitatum) using RAPD technology and successfully identified P. capitatum using specific SCAR fragments that were amplified with the constructed primers. A combination of RAPD and eastern blotting analyses using anti-ginsenoside Rb1 and Rg1 monoclonal antibodies by TANAKA et al [14] was used for the identification of P. notoginseng, P. quinquefolius and P. japonicas. JUNG et al [15] developed convenient and reliable
chloroplast genome-derived DNA markers for authentication of Korean and American ginseng in commercial processed products. The developed markers were successfully applied to evaluating the original species from various processed ginseng products purchased from markets in Korea and China. High-throughput application of this marker system will eradicate illegal trade and promote confident marketing for both species to increase the value of Korean as well as American ginseng in Korea and worldwide.
In the present study, RAPD was used to investigate genetic polymorphisms in three related herbs, P. ginseng, P. quinquefolium, and P. notoginseng. Subsequently, species-specific fragments were recovered, cloned and sequenced. The sequence data were used to design unique primers to develop a specific PCR-based identification method for P. ginseng. We then verified this method using several medicinal herb samples. The method reported here enabled the authentication of P. ginseng from other crude drugs such as P. quinquefolium, P. notoginseng and other Chinese traditional medical herbs. Our method has several advantages: 1) it is simple and costs- effective, 2) the results enable accurate identification of P. ginseng that is rapid, reliable and meets the needs of the market. 2 Materials and methods 2.1 Plant materials and DNA isolation
A total of 134 batches of samples were used in this study, including 33 batches of P. ginseng (Table 1), 14 batches of P. quinquefolium (Table 1), 19 batches of P. notoginseng (Table 1), 10 batches of adulterants (Table 2), and 58 batches of other traditional Chinese medicinal substances (Table 3). All samples were identified in the Hunan Institute for Drug Control and confirmed by our research group using DNA barcodes. Total genomic DNA was isolated using the CTAB method as described earlier [16].
2.2 RAPD analysis
A total of 120 RAPD primers were purchased from SBS Genetech Co. Ltd. (China). Two samples each of P. ginseng, P. quinquefolium and P. notoginseng were used for initial primer screening t o i d e n t i f y p r i m e r s t h a t p r o d u c e u n i q u e
J. Cent. South Univ. (2018) 25: 1052–1062
1054
Table 1 Information about P. ginseng, P. quinquefolium, and P. notoginseng samples
Specimen Species Origin/collect area Category Batch
R1 Panax ginseng NIFDC Reference herbal medicinal 1
R2-R3 P. ginseng Baishan, Jilin, China Cultivated ginseng 2
R4-R5 P. ginseng Baishan, Jilin, China Mountain cultivated ginseng 2
R6-R7 P. ginseng Benxi, Liaoning, China Mountain cultivated ginseng 2
R8-R12 P. ginseng Benxi, Liaoning, China Crude drugs 5
R13-R14 P. ginseng Fushun, Liaoning, China Mountain cultivated ginseng 2
R15 P. ginseng Fusong, Jilin, China Cultivated ginseng 1
R16 P. ginseng Fusong, Jilin, China Crude drugs 1
R17 P. ginseng Ji`an, Jilin, China Mountain cultivated ginseng 1
R18-R21 P. ginseng Ji`an, Jilin, China Cultivated ginseng 4
R22-R23 P. ginseng Jilin, China Crude drugs 2
R24-R26 P. ginseng Liaoning, China Crude drugs 3
R27-R28 P. ginseng Liaoning, China Cultivated ginseng 2
R29-R30 P. ginseng Tonghua, Jilin, China Cultivated ginseng 2
R31 P. ginseng Wangqing, Jilin, China Cultivated ginseng 1
R32 P. ginseng Xiaoxing`anling, Helongjiang, China Cultivated ginseng 1
R33 P. ginseng Yanji, Jilin, China Cultivated ginseng 1
X1 P. quinquefolium NIFD Reference herbal medicinal 1
X2 P. quinquefolium Huairou, Beijing, China Cultivated 1
X3-X5 P. quinquefolium Wendeng, Shandong, China Cultivated 3
X6 P. quinquefolium Changbaishan, Jinlin, China Cultivated 1
X7-X9 P. quinquefolium Canada Crude drugs 3
X10-X13 P. quinquefolium Jilin, China Crude drugs 3
X14 P. quinquefolium Shandong, China Crude drugs 1
S1 P.notoginseng NIFD Crude drugs 1
S2-S12 P.notoginseng Yunnan, China Crude drugs 11
S13 P.notoginseng Yanshan, Guangxi, China Cultivated 1
S14 P.notoginseng Malong, Yunnan, China Cultivated 1
S15 P.notoginseng Jianshui, Yunnan, China Cultivated 1
S16 P.notoginseng Napo, Guangxi, China Cultivated 1
S17 P.notoginseng Wenzhou, Guangxi, China Cultivated 1
S18 P.notoginseng Hongzhou, Yunnan, China Cultivated 1
S19 P.notoginseng Jinxi, Guangxi, China Cultivated 1
NIFD: National Institute for Food and Drug Control.
Table 2 Information about P. ginseng adulterant samples
No. Species Origin/collect area
H1 Sedum aizoon Huaihua, Hunan province, China
H2 Talinum paniculatum Lianqiao, Hunan province, China
H3 Codonopsis convolvulacea Kurz Lianqiao, Hunan province, China
H4 Schizocapsa plantaginea Hance Lianqiao, Hunan province, China
H5 Curcuma longa L. Leshan, Sichuan province, China
H6 Panacis japonici Rhizoma Lianqiao, Hunan province, China
H7 Phytolacca acinosa Roxb. Lianqiao, Hunan province, China
H8 Mirabilis jalapa L. Lianqiao, Hunan province, China
H9 Curcuma zedoaria (Christm.) Rosc. Lianqiao, Hunan province, China
H10 Wrightia laevis Lianqiao, Hunan province, China
J. Cent. South Univ. (2018) 25: 1052–1062
1055
Table 3 Information about other common traditional Chinese medicinal herb samples used in study
No. Species Origin/collect area
Z1 Polygonatum sibiricum Changsha, Hunan province, China
Z2 Magnolia officinalis Changsha, Hunan province, China
Z3 Eucommia ulmoides Oliv Changsha, Hunan province, China
Z4 Paeonia suffruticosa Andr. Changsha, Hunan province, China
Z5 Iris tectorum Maxim. Changsha, Hunan province, China
Z6 Citrus aurantium L. Changsha, Hunan province, China
Z7 Houttuynia cordata Changsha, Hunan province, China
Z8 Scrophularia ningpoensis Changsha, Hunan province, China
Z9 Peucedanum praeruptorum Changsha, Hunan province, China
Z10 Peucedanum decursivum Changsha, Hunan province, China
Z11 Citrus reticulata Blanco Changsha, Hunan province, China
Z12 Atractylodes chinensis Changsha, Hunan province, China
Z13 Polygonum multiflorum Changsha, Hunan province, China
Z14 Fritillaria cirrhosa Sichuan province, China
Z15 Polygala sibirica L. IMPLAD
Z16 Murraya exotica L. IMPLAD
Z17 Campsis radicans IMPLAD
Z18 Sauropus spatulifolius IMPLAD
Z19 Saussurea involucrata IMPLAD
Z20 Eriobotrya japonica IMPLAD
Z21 Leonurus japonicus IMPLAD
Z22 Euphorbia maculata L. IMPLAD
Z23 Lobelia chinensis Lour. IMPLAD
Z24 Polygala japonica IMPLAD
Z25 Eriocaulon buergerianum IMPLAD
Z26 Rabdosiae Rubescens IMPLAD
Z27 Murraya paniculata IMPLAD
Z28 Ilex cornuta Lindl IMPLAD
Z29 Potentilla chinensis IMPLAD
Z30 Juncus effusus L. IMPLAD
Z31 Selaginella pulvinata IMPLAD
Z32 Centella asiatica IMPLAD
Z33 Rhododendron mariesii IMPLAD
Z34 Strophanthus divaricatus IMPLAD
Z35 Trachelospermum jasminoides IMPLAD
Z36 Ilex rotunda IMPLAD
Z37 Cynanchum stauntonii IMPLAD
Z38 Zanthoxylum schinifolium IMPLAD
Z39 Impatiens balsamina IMPLAD
Z40 Cannabis sativa L. IMPLAD
Z41 Trigonella foenum-graecum L. IMPLAD
Z42 Perilla frutescens. IMPLAD
To be continued
J. Cent. South Univ. (2018) 25: 1052–1062
1056
Continued No. Species Origin/collect area
Z43 Allium tuberosum IMPLAD
Z44 Nelumbo nucifera Gaertn. IMPLAD
Z45 Zanthoxylum bungeanum IMPLAD
Z46 Astragalus complanatus IMPLAD
Z47 Glycyrrhizae glabra IMPLAD
Z48 Aesculus chinensis IMPLAD
Z49 Aesculus wilsonii Rehd. IMPLAD
Z50 Menispermi Rhizoma IMPLAD
Z51 Bupleurum chinense DC IMPLAD
Z52 Benincasa hispida IMPLAD
Z53 Acanthopanax gracilistylu IMPLAD
Z54 Platycodon grandiflorum IMPLAD
Z55 Polygala tenuifolia Willd. IMPLAD
Z56 Astragalus membranaceus IMPLAD
Z57 Glycyrrhiza uralensis IMPLAD
Z58 Astragalus membranaceus IMPLAD
IMPLAD: Institute of Medicinal Plant Development, Beijing, China.
amplification bands. The identified primers were then confirmed by screening a larger number of samples. Each random primer was used for amplification at least twice. RAPD analysis was performed as previously described [15]. 2.3 Cloning, sequencing and designing SCAR
primers The RAPD band specific to P. ginseng was
cloned into the pGM-T vector. The positive plasmids were bi-directionally sequenced by Biosune Biotech Co. Ltd. (China) using the universal primer pair T7/SP6. To ensure the accuracy of the sequences, at least two clones were sequenced [17, 18]. The sequences were assembled and edited using DNAStar 4.05 software (DNAStar, Inc.). The nucleotide sequence of the fragment C-12 was deposited in Genbank and primers to test P. ginseng specific to the identified sequences were designed using the primer-design software Oligo 6. 2.4 Development and optimization of PCR-based
identification method for P. ginseng The PCR reaction mixture consisted of 5.0 μL
of 5× Primer STAR Buffer (Mg2+ plus), 2.5 μL of dNTPs (2.5 mmol/L), 0.75 μL of each of the designed primers: F1 and R1 (10 μmol/L), 0.25 μL of PrimeSTAR HS DNA polymerase (2.5 U/μL, Takara, China), and 1.0 μL of DNA template. The
final volume of the reaction mixture was adjusted to 25 μL with sterilized ddH2O.
The amplification conditions were as follows: initial denaturation at 95 °C for 3 min, 30 cycles of denaturation at 98 °C for 10 s, annealing at 52 °C for 15 s, extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. The reactions were performed in a Veriti PCR thermal cycler (Applied Biosystems, USA).
This study also investigated the effect of different annealing temperatures, numbers of amplification cycles, amounts of DNA template, and types of Taq enzyme on the performance of the PCR-based identification method. Sterilized ddH2O was used as a blank control. Additionally, the universal primer pair ITS2 (ITS2F: ATGCGATACTTGGTGTGAAT; ITS3R: GACGCTTCTCCAGACTACAAT) was used to perform DNA amplification of all samples to be tested to verify the reliability of the template DNA. 2.5 Verification of P. ginseng-specific SCAR
marker DNA samples from 134 medicinal herb
samples (Tables 1–3) were used as templates for P. ginseng-specific PCR amplification. The details of the reaction system, reaction conditions, and detection method were the same as those described in Section 2.4.
J. Cent. South Univ. (2018) 25: 1052–1062
1057
3 Results and discussion 3.1 Screening for P. ginseng-specific RAPD
markers RAPD amplification assays performed using
120 random primers revealed that random primers A-12, B-05, C-07, C-12, F-07, and F-08 (Table 4) produced unique amplification products (Figure 1). Of these, random primers A-12 (Figure 1(a)) and B-05 (Figure 1(b)) produced unique bands in P. notoginseng, random primers C-07 (Figure 1(c)) and F-08 (Figure 1(d)) produced unique bands in P. quinquefolium, and random primers F-07 (Figure 1(e)) and C-12 (Figure 1(f)) produced unique bands in P. ginseng. Figure 1(f) shows the results of RAPD amplification using the random primer C-12, with a P. ginseng specific RAPD marker C-12 at approximately 600 bp, and no markers for the P. quinquefolium and P. notoginseng samples, highlighting the specificity of the primers. Table 4 Random primers for RAPD analysis
No. Sequence
A-12 TCGGCGATAG
C-12 TGTCATCCCC
B-05 TGCGCCCTTC
F-07 CCGATATCCC
C-07 GTCCCGACGA
F-08 GGGATATCGG
3.2 Conversion to SCAR markers
The six identified unique RAPD fragments were recovered from gels, purified and cloned into TA vectors. The clones obtained were sequenced using universal primers, and the corresponding nucleotide sequences were obtained. To confirm whether the RAPD marker C-12 is specific for P. ginseng, its nucleotide sequence was subjected to BlastN homology search against the nonredundant GenBank database. The search results showed no significant match with any previously determined sequences. The nucleotide sequence of the P. ginseng-specific marker C-12 has been submitted to GenBank with the accession number KU553472. Finally, an identification primer pair (F1: 5’CCCGACTCAAAATCGAAGT3’, R1: 5’AAGCAAGAAGCAAGGGTAACATA3’; Chinese Patent Application Number: 201510288554.0) was
designed based on the obtained sequence information using Oligo 6 primer design software. 3.3 Development and optimization of SCAR
identification method specific for P. ginseng The primers F1 and R1 were subsequently
used to develop the SCAR identification method specific for P. ginseng. As expected, the amplification of P. ginseng produced a band of approximately 300 bp in size, which also appeared in P. quinquefolius and P. notoginseng. Interestingly, a band at approximately 130 bp was observed only in P. ginseng but was absent among P. quinquefolius and P. notoginseng amplicons. These results suggested that this unique band pattern might distinguish P. ginseng from P. quinquefolius and P. notoginseng (Figure 2). To investigate the specificity and stability of this band pattern, it was necessary to study the effect of the annealing temperature, the number of cycles, the amount of template DNA, and the fidelity of the DNA polymerase used.
To determine the most suitable annealing temperature for amplification, various annealing temperatures (52, 54, 56 and 58 °C) were tested. The results demonstrated that at an annealing temperature of 52–58 °C, amplification of all P. ginseng samples generated two unique fragments at approximately 300 bp and 130 bp (Figure 2(a)). Considering the effects of annealing temperature on primer amplification efficiency and specificity, we selected 56 °C as the ideal annealing temperature.
Various numbers of amplification cycles (30, 33, 36 and 39) were tested to determine the optimum number of amplification cycles. The results showed that 30 cycles were sufficient to produce a satisfactory intensity of bands (Figure 2(b)). To minimize the possibility of appearance of non-specific bands caused by excessive amplification cycles, 30 was selected as an ideal number of cycles for the PCR assay to ensure the accuracy of results.
To optimize the amount of DNA to be used as a template for the PCR assay, the DNA concentration was adjusted to approximately 30 ng/μL and various volumes of DNA (0.2, 1, 2 and 3 μL) were used in 25-μL PCR reactions. The results indicated that satisfactory PCR amplification was obtained with 0.2–3 μL of DNA template (equivalent to 6–90 ng of total DNA). It was
J. Cent. South Univ. (2018) 25: 1052–1062
1058
Figure 1 RAPD profiles of P. ginseng, P. quinquefolius and P. notoginseng amplified by primer: (a) A-12; (b) B-05;
(c) C-07; (d) F-08; (e) F-07; (f) C-12 (Lanes 1 through 10 are P. Ginseng samples R2 through R11 in Table 1,
respectively. Lanes 11 to 20 are P. quinquefolius samples X2 to X11 in Table 1, respectively. Lanes 21–29 are P.
Notoginseng samples S2 to S10 in Table 1, respectively. M is the DNA marker, indicating DNA sizes of 2000 bp, 1000
bp, 750 bp, 500 bp, 250 bp, and 100 bp, from top to bottom. The arrow indicates the polymorphic band)
observed that at sufficient number of cycles (30 cycles), the amplified bands became brighter with increasing amount of template DNA, with no effect
on the results (Figure 2(c)). To establish the effect of DNA polymerases
wi th d i f fe ren t f ide l i t i es on ampl i f ica t ion ,
J. Cent. South Univ. (2018) 25: 1052–1062
1059
Figure 2 Optimization of PCR-based identification method for P. Ginseng: (a) Effect of annealing temperature on
identification results; (b) Results of PCR amplification with different numbers of cycles; (c) Results of PCR
amplification using different amounts of DNA template; (d) Effect of fidelity of DNA polymerase on specificity of
SCAR analysis (Lanes 1 and 2 are sample R2 and R6 of P. ginseng (Table 1), respectively. Lanes 3 and 4 are sample X2
and X3, P. quinquefolium (Table 1), respectively. Lanes 5 and 6 are Sample S2 and S3 of P. notoginseng (Table 1),
respectively. Lane 7 is adulterants Mirabilis jalapa L. Lane 8 is the negative control. M is the DNA 50 bp marker
(Tiangen), indicating DNA sizes of 500 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, and 50 bp, from
top to bottom)
high-fidelity HS DNA polymerase with regular buffer (Takara), high-fidelity HS DNA polymerase with GC buffer (Takara), high-fidelity PCR Master (Roche), Phanta Super-Fidelity DNA polymerase (Vazyme), and regular Taq enzyme (Tiangen) were used for PCR amplification in separate reactions. Specific amplification bands were observed when HS DNA polymerase (Takara) was used. The results also showed that with the same HS DNA polymerase, different buffers resulted in significantly different intensities of the amplified
bands (Figure 2(d)). Overall, high-fidelity PrimeSTAR HS DNA polymerase with regular buffer (Takara) demonstrated the best performance. 3.4 Verification of SCAR markers
The optimized PCR-based identification system for P. ginseng was further evaluated by testing the 134 batches of samples. In this study, specific PCR products were produced from these samples (Tables 1–3) after amplification using ITS2 universal primers, suggesting that the quality of
J. Cent. South Univ. (2018) 25: 1052–1062
1060
DNA from the samples met the assay requirements. The electropherogram (Figure 3(a)) revealed that amplification of all P. ginseng samples produced two unique bands at approximately 300 bp and 130 bp. As expected, this band pattern was absent in P. quinquefolium (Figure 3(b)), P. notoginseng
(Figure 3(c)), Sedum aizoon, Talinum paniculatum, Codonopsis convolvulacea Kurz, Schizocapsa plantaginea Hance, Curcuma longa L., Panacis Japonici Rhizoma, Phytolacca acinosa Roxb. (Figure 3(c)), and 58 other traditional Chinese medicinal substances studied (Figure 3(e)). These
Figure 3 SCAR analysis of P. ginseng and their adulterants amplified by specific primers F1 and R1: (a) Lanes 1 to 33
are samples R1 to R33 of P. ginseng (Table 1), respectively; (b) Lane 1 is Sample R1 of P. ginseng. Lanes 2 throgh 15
are samples X1 through X14 of P. quinquefolium (Table 1), respectively; (c) Lane 1 is sample R1 of P. ginseng. Lanes 2
to 20 are samples S1 to S19 of P. notoginseng (Table 1), respectively; (d) Lanes 1 to 10 are samples Sedum aizoon,
Talinum paniculatum, Codonopsis convolvulacea Kurz, Schizocapsa plantaginea Hance, Curcuma longa L., Panacis
Japonici Rhizoma, Phytolacca acinosa Roxb., Mirabilis jalapa L., Curcuma zedoaria (Christm.) Rosc., and Wrightia
laevis (Table 2), respectively (Lane 11 is the blank control. Lane 12 is Sample R1 of P. Ginseng); (e) Lane 1 denotes
Sample R1 of P. ginseng. Lanes 2 through 59 correspond to the 58 Chinese medicinal herbs in Table 3, e.g., from Z1
(Polygonatum sibiricum) and Z2 (Magnolia officinalis) to Z58 (Astragalus membranaceus). Lane 60 is the negative
control. M is the DNA 50 bp marker (Tiangen), indicating DNA sizes of 500 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp,
150 bp, 100 bp, and 50 bp, from top to bottom
J. Cent. South Univ. (2018) 25: 1052–1062
1061
results indicated that the identified ginseng-specific SCAR marker could be used to specifically distinguish P. ginseng from other medicinal herbs. 4 Conclusions
In this study, we successfully developed a SCAR assay to identify P. ginseng among traditional Chinese medicinal products. Our achievements can be summarized as follows:
1) Random primers A-12, B-05, C-07, C-12, F-07, and F-08 produced unique amplification products in P. ginseng, P. quinquefolium, and P. notoginseng in RAPD amplification assays.
2) The RAPD marker was converted to a stable SCAR marker, by designing a P. ginseng-specific identification primer pair (F1: 5’CCCGACTCAAAATCGAAGT3’, R1: 5’AAGCAAGAAGCAAGGGTAACATA3’; Chinese Patent Application Number: 201510288554.0).
3) The SCAR marker-based identification system for Panax ginseng was developed, after optimization of the reaction conditions, which generated a unique band pattern (two DNA fragments at approximately 300 bp and 130 bp), only in P. ginseng samples. This pattern was not observed upon amplification of P. quinquefolium, P. notoginseng, Sedum aizoon, Talinum paniculatum, Codonopsis convolvulacea Kurz, Schizocapsa plantaginea Hance, Curcuma longa L., Panacis Japonici Rhizoma, Phytolacca acinosa Roxb, and 58 other medicinal herb samples examined in this study. The observed results confirmed that the proposed method could be used to authenticate P. ginseng. The proposed method adopts a PCR-based specific SCAR marker technology to identify ginseng and its decoction products and involves a series of simple processes of DNA extraction, PCR amplification, and electrophoresis. Owing to advantages such as easy operation, high specificity, and requirement of a small sample size, this method is a practical and feasible tool for P. ginseng identification, with many potential applications. The SCAR analysis is expected to be particularly relevant for the establishment of reliable DNA fingerprinting for quality control of P. ginseng.
References [1] ATTELE A S, WU Ji-an, YUAN Chun-su. Ginseng
pharmacology: Multiple constituents and multiple actions [J].
Biochemical Pharmacology, 1999, 58(11): 1685–1693.
[2] CHANG Y S, SEO E K, GYLLENHAAL C, BLOCK K I.
Panax ginseng: a role in cancer therapy? [J]. Integrative
Cancer Therapies, 2003, 2(1): 13–33.
[3] CHENG Yong, SHEN Li-hong, ZHANG Jun-tian.
Anti-amnestic and anti-aging effects of ginsenoside Rg1 and
Rb1 and its mechanism of action [J]. Acta Pharmacologica
Sinica, 2005, 26(2): 143–149.
[4] CHOO M K, PARK E K, HAN M J, KIM D H. Antiallergic
activity of ginseng and its ginsenosides [J]. Planta Medica,
2003, 69(6): 518–522.
[5] SHIN H R, KIM J Y, YUN T K, MORGAN G, VAINIO H.
The cancer-preventive potential of Panax ginseng: A review
of human and experimental evidence [J]. Cancer Causes &
Control, 2000, 11(6): 565–576.
[6] WANG Hong-wei, PENG Da-cheng, XIE Jing-tian. Ginseng
leaf-stem: Bioactive constituents and pharmacological
functions [J]. Chinese Medicine, 2009, 4(1): 20. DOI:
https://doi.org/10.1186/1749-8546-4-20.
[7] HE Yan-qing. The discuss ginseng market situation and the
authenticity of identification [J]. China Practical Medical,
2011, 6(18): 226–227.
[8] WU Xue-ling, LIU Li-li, ZHANG Zhen-zhen, DENG
Fan-fan, LIU Xin-xing. Molecular characterization of
Acidithiobacillus ferrooxidans strains isolated from different
environments by three PCR-based methods [J]. Journal of
Central South University, 2015, 22(4): 455−465.
[9] CHEN Li-jing, QI Xin, WANG Yu-kun, ZHANG Li, GUO
Zhi-fu, LIN Jing-wei, SONG Yu-ning, ZHONG Ming.
Identification of Schisandra sphenanthera and S. chinensis by
random amplified polymorphic DNA sequence characterized
applied region] [J]. China Journal of Chinese Materia
Medica, 2011, 36(22): 3083–3085.
[10] CUI Zhan-hu, LONG Ping, WANG Ying-li, BAI Xiao-rong,
YUAN Yuan, LI Min-hui. Application and prospect of DNA
molecular markers in the identification of Chinese Medicine
[J]. Journal of Chinese Medicinal Materials, 2015, 38(1):
188–192.
[11] KIRAN U, KHAN S, MIRZA K J, RAM M, ABDIN M Z.
SCAR markers: A potential tool for authentication of herbal
drugs [J]. Fitoterapia, 2010, 81(8): 969–976.
[12] SUN Tao, TENG Shao-na, KONG De-ying, SONG Yun, XU
Jin, LI Ying-guo, WANG Yu, LI Ming-fu. DNA barcoding
used in the identification of ginseng [J]. China
Biotechnology, 2013, 33(4): 143–148.
[13] ZHOU Tao, XIE Yu, ZHANG Li-yan, WEI Sheng-hua, JIN
Yan-lei. Study on sequence characterized amplified region
(SCAR) markers of polygonum capitatum [J]. China Journal
of Chinese Materia Medica, 2013, 38(16): 2577–2580.
[14] TANAKA H, FUKUDA N, SHOYAMA Y. Identification and
differentiation of Panax species using ELISA, RAPD and
eastern blotting [J]. Phytochemical Analysis, 2006, 17(1):
46–55.
[15] JUNG Juyeon, KIM Kyung-hee, YANG Kiwoung, BANG
Kyong-hwan, YANG Tae-Jin. Practical application of DNA
markers for high-throughput authentication of Panax ginseng
J. Cent. South Univ. (2018) 25: 1052–1062
1062
and Panax quinquefolius from commercial ginseng products
[J]. Journal of Ginseng Research, 2014, 38(2): 123–129.
[16] PIRTTILÄ A M, HIRSIKORPI, M, KÄMÄRÄINEN T,
JAAKOLA L, HOHTOLA A. DNA isolation methods for
medicinal and aromatic plants [J]. Plant Molecular Biology
Reporter, 2001, 19(3): 273–273.
[17] SOLIERI L, GIUDICI P. Development of a sequence-
characterized amplified region marker-targeted quantitative
PCR assay for strain-specific detection of Oenococcus oeni
during wine malolactic fermentation [J]. Applied and
Environmental Microbiology, 2010, 76(23): 7765–7774.
[18] XIA Jin-lan, ZHANG Qian, ZHANG Rui-yong, PENG
Juan-hua, PENG An-an, ZHAO Xiao-juan, NIE Zhen-yuan,
QIU Guan-zhou. Isolation and characterization of acidophilic
bacterium from Dongxiangshan Mine in Xinjiang Province,
China [J]. Journal of Central South University, 2010, 17(1):
50−55.
(Edited by YANG Hua)
中文导读
用于药品检验的人参 SCAR 标记研究 摘要:为了研究人参、西洋参和三七的基因多态性,使用 120 条随机引物进行了随机扩增多态性 DNA分析。将筛选获得的人参特异性 RAPD 标记 C-12 进行 T-A 克隆、测序(GenBank 登录号 KU553472)。根据序列分析结果,设计了一对特异性引物,经优化反应条件,建立了人参的 SCAR 标记鉴别体系。
在所有的 33 批人参标本中,均能稳定地获得约 300 bp 和 130 bp 的 2 条阳性扩增带;在混伪品以及西
洋参、三七和其他中药材共计 101 批标本中均为阴性扩增。因此,建立了一种快速、有效的能准确、
特异性地鉴别人参的 PCR 方法,可用于人参的种质资源保护、利用及其基原鉴定。 关键词:人参;随机扩增多态性 DNA(RAPD);特征序列扩增标记(SCAR);分子鉴定
Recommended