Planta Med 2008; 74(4): 464-469
DOI: 10.1055/s-2008-1034325
Biochemistry and Molecular Biology
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

Rapid Authentication of Bupleurum Species using an Array of Immobilized Sequence-Specific Oligonucleotide Probes

Wen-Yu Lin1 , Li-Ru Chen1 , Tsai-Yun Lin1
  • 1Institute of Bioinformatics and Structural Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China
Further Information

Publication History

Received: May 14, 2007 Revised: November 13, 2007

Accepted: January 26, 2008

Publication Date:
11 March 2008 (online)

Abstract

Standardization and modernization of Chinese medicinal herbs are limited partially by misidentification of processed materials. Our goal was to develop an efficient method for verification of Chinese medicinal herbs, based on the variable sites of the rDNA internal transcribed spacer (ITS) region. We analyzed sequence differences in ITS of three Bupleurum species, B. kaoi Liu Chao et Chuang, B. falcatum L. and B. chinense DC., and developed a rapid detection method using a sequence-specific oligonucleotide probe (SSOP) array. The SSOP array, composed of poly-T tailed sequence-specific oligonucleotides, was hybridized to the digoxigenin (DIG)-labeled target ITS DNA of the Bupleurum species. The detected signals corresponded precisely to the specific sequences. This array provides a reliable and economical method for authenticating a large number of Chinese medicinal herbs. The short duration of the procedure (within 30 h) makes it an especially useful tool in verifying processed plant material.

Abbreviations

CSPD:disodium 3-{4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclodecan]-4-yl}phenyl phosphate

DIG:digoxigenin

GCG:genetics computer group

ITS:inernal transcribed spacer

PCR:polymerase chain reaction

rDNA:ribosomal DNA

RFLP:restriction fragment length polymorphism

SSPO:sequence-specific oligonucleotide probe

TdT:terminal deoxyribonucleotidyl transferase

Tm:melting temperature

References

  • 1 Navarro P, Giner R M, Recio M C, Manez S, Cerda-Nicolas M, Rios J L. In vivo anti-inflammatory activity of saponins from Bupleurum rotundifolium. .  Life Sci. 2001;  68 1199-206
  • 2 Yen M H, Weng T C, Liu S Y, Chai C Y, Lin C C. The hepatoprotective effect of Bupleurum kaoi, an endemic plant to Taiwan, against dimethylnitrosamine-induced hepatic fibrosis in rats.  Biol Pharm Bull. 2005;  28 442-8
  • 3 Neves S S, Watson M F. Phylogenetic relationship in Bupleurum (Apiaceae) based on nuclear ribosomal DNA ITS sequence data.  Ann Bot. 2004;  93 379-98
  • 4 Zhao Z, Hu Y, Liang Z, Yuen J PS, Jiang Z, Leung K SY. Authentication is fundamental for standardization of Chinese medicines.  Planta Med. 2006;  72 865-74
  • 5 Hon C C, Chow Y C, Zeng F Y, Leung F CC. Genetic authentication of ginseng and other traditional Chinese medicine.  Acta Pharmacol Sin. 2003;  24 841-6
  • 6 Zhang Y B, Wang J, Wang Z T, But P P, Shaw P C. DNA microarray for identification of the herb of Dendrobium species from Chinese medicinal formulations.  Planta Med. 2003;  69 1172-4
  • 7 Lau D TW, Shaw P C, Wang J, But P PH. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA.  Planta Med. 2001;  67 456-60
  • 8 Conner B J, Reyes A A, Morin C, Itakura K, Teplitz R L, Wallace R B. Detection of sickle cell βS-globin allele by hybridization with synthetic oligonucleotides.  Proc Natl Acad Sci USA. 1983;  80 278-82
  • 9 Rogers S O, Bendich A J. Extraction of DNA from plant tissues. In: Gelvin SB, Schilperoot RA, Verma DPS, editors. Plant molecular biology manual.  Dordrecht: Kluywer Academic. Publishers;  1988 A6 1-10
  • 10 Ngan F, Shaw P, But P, Wang J. Molecular authentication of Panax species.  Phytochemistry. 1999;  50 789-91
  • 11 Lee S B, Rasmussen S K. Molecular markers in some medicinal plants of the Apiaceae family.  Euphytica. 2000;  114 87-91

Prof. Dr. Tsai-Yun Lin

Institute of Bioinformatics and Structural Biology and Department of Life Science

National Tsing Hua University

Hsinchu 30013

Taiwan

Republic of China

Phone: +886-3-574-2758

Fax: +886-3-571-5934

Email: tylin@life.nthu.edu.tw