Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant and Herbal Materials
2.2. Preparation of Genomic DNA and PCR Amplification
2.3. Analysis of Nucleotide Sequences and Phylogenetic Relationships
2.4. Development of the Multiplexed SCAR Marker Assay and Monitoring of Commercial Herbal Medicines
3. Results
3.1. Analysis of nrDNA-ITS Sequences
3.2. Development of SCAR Markers for Distinguishing Herbal Medicines
3.3. Establishment of a Multiplex SCAR Amplification Method and Monitoring of Commercially-Processed Herbal Medicines
4. Discussion
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Name | Habitat Information | Voucher Number | Abbreviation | Lane in Gel | |
---|---|---|---|---|---|
Scientific Name (Family) | Herbal Name | ||||
Adenophora stricta Miq. (Campanulaceae) | Adenophorae Radix | Caoping, Li, Longnan, Gansu, China | KIOM200701000519 | AS-CP | 1 |
Jisan, Jindo, Jeonnam, Korea | KIOM200701000873 | AS-JS | 2 | ||
Guyuan, Ningxia, China | KIOM201201005303 | AS-GA | 3 | ||
Sangju, Gyeongbuk, Korea | KIOM201301006488 | AS-SJ | 4 | ||
Adenophora triphylla (Thunb.) A. DC. (Campanulaceae) | Adenophorae Radix | Cheoncheon, Jangsu, Jeonbuk, Korea | KIOM200701000682 | AT-CC | 5 |
Yaksan, Wando, Jeonnam, Korea | KIOM200701000741 | AT-YS | 6 | ||
Daegang, Danyang, Chungbuk, Korea | KIOM201101004148 | AT-DG | 7 | ||
Longjing, Yanbian, Jilin, China | KIOM201201005563 | AT-LJ | 8 | ||
Adenophora triphylla var. japonica (Regel) Hara (Campanulaceae) | Adenophorae Radix | Sancheong, Gyeongnam, Korea | KIOM201201004951 | ATJ-SC | 9 |
Beichuan, Mianyang, Sichuan, China | KIOM201201005359 | ATJ-BC | 10 | ||
Sangju, Gyeongbuk, Korea | KIOM201301006312 | ATJ-SJ | 11 | ||
Gandong, Hwacheon, Gangwon, Korea | KIOM201401009347 | ATJ-GD | 12 | ||
Codonopsis lanceolata (Siebold & Zucc.) Benth. & Hook. f. ex Trautv. (Campanulaceae) | Codonopsis Lanceolatae Radix | Sangju, Namhae, Gyeongnam, Korea | KIOM201001002725 | CL-SJ | 13 |
Sinan, Jeonnam, Korea | KIOM201101003615 | CL-SN | 14 | ||
Geochang, Gyeongnam, Korea | KIOM201201004752 | CL-GC | 15 | ||
Codonopsis pilosula (Franch.) Nannf. (Campanulaceae) | Codonopsis Pilosulae Radix | Ya’an, Sichuan, China | KIOM201101004238 | CP-YA | 16 |
Helong, Yanbian, Jilin, China | KIOM201101004335 | CP-HL | 17 | ||
Girin, Inje, Gangwon, Korea | KIOM200701000339 | CP-GR | 18 | ||
Jinbu, Pyeongchang, Gangwon, Korea | KIOM200801001104 | CP-JB | 19 | ||
Wangqing, Yanbian, Jilin, China | KIOM201101004270 | CP-WQ | 20 | ||
Glehnia littoralis F. Schmidt ex Miq. (Apiaceae) | Glehniae Radix | Gujwa, Jeju, Jeju, Korea | KIOM200601000041 | GL-JJ | 21 |
Anmyeon, Taean, Chungnam, Korea | KIOM200701000230 | GL-AM | 22 | ||
Geojin, Goseong, Gangwon, Korea | KIOM200701000231 | GL-GJ | 23 | ||
Jukbyeon, Uljin, Gyeongbuk, Korea | KIOM200601000062 | GL-JB | 24 |
Species | Sequence Length (bp) | Intra-Species Variability (%) | Inter-Species Variability (%) | |||
---|---|---|---|---|---|---|
Constant | Aligned | ITS1 | ITS2 | ITS1 | ITS2 | |
A. stricta | 793–795 | 799 | 0.0205 ± 0.0104 | 0.0198 ± 0.0114 | 0.2840 ± 0.2322 | 0.2657 ± 0.2132 |
A. triphylla | 793–795 | 799 | 0.0111 ± 0.0062 | 0.0198 ± 0.0101 | 0.2760 ± 0.2279 | 0.2661 ± 0.2169 |
A. triphylla var. japonica | 793–795 | 799 | 0.0092 ± 0.0061 | 0.0192 ± 0.0094 | 0.2778 ± 0.2292 | 0.2686 ± 0.2184 |
C. lanceolata | 743 | 799 | 0.0000 ± 0.0000 | 0.0029 ± 0.0025 | 0.3566 ± 0.1824 | 0.3233 ± 0.1893 |
C. pilosula | 743 | 799 | 0.0047 ± 0.0061 | 0.0035 ± 0.0045 | 0.3480 ± 0.1560 | 0.3375 ± 0.1678 |
G. littoralis | 689 | 799 | 0.0023 ± 0.0025 | 0.0000 ± 0.0000 | 0.5810 ± 0.0122 | 0.5717 ± 0.0133 |
Primer Direction | Primer Name | Primer Sequence (5′ → 3′) | Specificity | Amplicon Size (bp) |
---|---|---|---|---|
Forward | AD ITS1 SCAR F | ATC GAG CGA AAG CGC GTG AGC T | Adenophora sp. | 231 |
CL ITS1 SCAR F | TGG CCC CTT GCC GTC GAC CT | C. lanceolata | 267 | |
CP ITS1 SCAR F | AAA ACT TAA CTC AAA GAG CGC GA | C. pilosula | 167 | |
GL ITS1 SCAR F | GTA CGT CCG TAT CCC GTT AAG G | G. littoralis | 124 | |
Reverse | ITS1 SCAR R | GCA ATT CAC ACC AAG TAT CGC AT | All samples | - |
No. | Voucher No. | Product Name | Multiplex-SCAR Result | Country |
---|---|---|---|---|
1 | 2-15-0678 | Adenophorae Radix | Adenophorae Radix | Korea |
2 | 2-15-0679 | Adenophorae Radix | Adenophorae Radix | Korea |
3 | 2-15-0680 | Adenophorae Radix | Adenophorae Radix | Korea |
4 | 2-15-0681 | Adenophorae Radix | Adenophorae Radix | Korea |
5 | 2-15-0682 | Adenophorae Radix | Adenophorae Radix | Korea |
6 | 2-15-0683 | Adenophorae Radix | Adenophorae Radix | Korea |
7 | 2-15-0684 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
8 | 2-15-0685 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
9 | 2-15-0705 | Adenophorae Radix | Adenophorae Radix | China |
10 | 2-15-0706 | Adenophorae Radix | Adenophorae Radix | China |
11 | 2-15-0707 | Adenophorae Radix | Adenophorae Radix | China |
12 | 2-15-0708 | Adenophorae Radix | Adenophorae Radix | China |
13 | 2-15-0711 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
14 | 2-15-0712 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
15 | 2-15-0713 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
16 | 2-15-0714 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
17 | 2-15-0715 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
18 | 2-15-0716 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
19 | 2-15-0717 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
20 | 2-15-0718 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
21 | 2-15-0719 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
22 | 2-15-0720 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
23 | 2-15-0721 | Codonopsis Lanceolatae Radix | Codonopsis Lanceolatae Radix | Korea |
24 | 2-15-0722 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
25 | 2-15-0723 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
26 | 2-16-0302 | Adenophorae Radix | Codonopsis Lanceolatae Radix | Korea |
27 | 2-15-0730 | Glehniae Radix | Glehniae Radix | Korea |
28 | 2-15-0731 | Glehniae Radix | Glehniae Radix | Korea |
29 | 2-15-0732 | Glehniae Radix | Glehniae Radix | Korea |
30 | 2-15-0733 | Glehniae Radix | Glehniae Radix | Korea |
31 | 2-15-0734 | Glehniae Radix | Glehniae Radix | Korea |
32 | 2-15-0735 | Glehniae Radix | Glehniae Radix | Korea |
33 | 2-15-0736 | Glehniae Radix | Glehniae Radix | Korea |
34 | 2-15-0737 | Glehniae Radix | Glehniae Radix | Korea |
35 | 2-17-0017 | Codonopsis Pilosulae Radix | Codonopsis Pilosulae Radix | China |
36 | 2-16-0025 | Codonopsis Pilosulae Radix | Codonopsis Pilosulae Radix | China |
37 | 2-15-0025 | Codonopsis Pilosulae Radix | Codonopsis Pilosulae Radix | China |
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Moon, B.C.; Kim, W.J.; Han, K.S.; Yang, S.; Kang, Y.; Park, I.; Piao, R. Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers. Appl. Sci. 2017, 7, 660. https://doi.org/10.3390/app7070660
Moon BC, Kim WJ, Han KS, Yang S, Kang Y, Park I, Piao R. Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers. Applied Sciences. 2017; 7(7):660. https://doi.org/10.3390/app7070660
Chicago/Turabian StyleMoon, Byeong Cheol, Wook Jin Kim, Kyeong Suk Han, Sungyu Yang, Youngmin Kang, Inkyu Park, and Renzhe Piao. 2017. "Differentiating Authentic Adenophorae Radix from Its Adulterants in Commercially-Processed Samples Using Multiplexed ITS Sequence-Based SCAR Markers" Applied Sciences 7, no. 7: 660. https://doi.org/10.3390/app7070660