Authentication of Medicinal Herbs using PCR-Amplified ITS2 with Specific Primers

Abstract

Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.

Abbreviations

ITS:internal transcribed spacer

ITS1:internal transcribed spacer 1

ITS2:internal transcribed spacer 2

Key words

Authentication - Aristolochiaceae - Aristolochia fangchi - Aristolochia manshuriensis - Clematis argentilucida - internal transcribed spacer - Menispermaceae - Ranunculaceae - Stephania tetrandra

References

• 1 Shaw P C, Wang J, But P PH. Authentication of Chinese medicinal materials by DNA technology, 1st edition. Hackensack; World Scientific Publishing 2002: 229-31
• 2 Lau D T, 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.
• 3 Zhao Z L, Zhou K Y, Dong H, Xu L S. Characters of nrDNA ITS region sequences of fruits of Alpinia galanga and their adulterants.  Planta Med. 2001;  67 381-3.
• 4 Shiba M, Kondo K, Miki E, Yamaji H, Morota T, Terabayashi S. et al . Identification of medicinal Atractylodes based on ITS sequences of nrDNA.  Biol Pharm Bull. 2006;  29 315-20.
• 5 Cubero O F, Crespo A, Fatehi J, Bridge P D. DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi.  Plant Syst Evol. 1999;  216 243-9.
• 6 Bailey C D, Carr T G, Harris S A, Hughes C E. Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes.  Mol Phylogenet Evol. 2003;  29 435-55.
• 7 Baldwin B G, Sanderson M J, Porter J M, Wojciechowski M F, Campbell C S, Donoghue M J. The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny.  Ann Missouri Bot Gard. 1995;  82 247-77.
• 8 Ngan F N, Shaw P C, But P PH, Wang J. Molecular authentication of Panax species.  Phytochem. 1999;  50 787-91.
• 9 Fu R Z, Wang J, Zhang Y B, Wang Z T, But P PH, Li N. et al . Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction-restriction fragment length polymorphism.  Planta Med. 1999;  67 456-60.
• 10 Hillis D M, Dixon M T. Ribosomal DNA: Molecular evolution and phylogenetic interference.  Q Rev Biol. 1991;  66 411-53.
• 11 LeRoy A, Potter E, Woo H H, Heber D, Hirsch A M. Characterization and identification of alfalfa and red clover dietary supplements using a PCR-based method.  J Agric Food Chem. 2002;  50 5063-9.
• 12 Zhao Z, Hu Y, Liang Z, Yuen J P, Jiang Z, Leung K S. Authentication is fundamental for standardization of Chinese medicines.  Planta Med. 2006;  72 865-74.
• 13 Alvarez I, Wendel J F. Ribosomal ITS sequences and plant phylogenetic inference.  Mol Phylogenet Evol. 2003;  29 417-34.
• 14 Liu J S, Schardl C L. A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes.  Plant Mol Biol. 1994;  26 775-8.
• 15 Jobes D V, Thien L B. A conserved motif in the 5.8S ribosomal RNA (rRNA) gene is a useful diagnostic marker for plant internal transcribed spacer (ITS) sequences.  Plant Mol Biol Rep. 1997;  15 326-34.
• 16 Stiller J W, McClanahan A. Phyto-specific 16S rDNA PCR primers for recovering algal and plant sequences from mixed samples.  Mol Ecol Notes. 2005;  5 1-3.
• 17 Kitano T, Umetsu K, Tian W, Osawa M. Two universal primer sets for species identification among vertebrates. Int J Legal Med online publication 15 July 2006: doi: 10.1007/s00414 - 006 - 0113-y.