Species Identification from Ginseng Drugs by Multiplex Amplification Refractory Mutation System (MARMS)

Abstract

The multiplex amplification refractory mutation system (MARMS) was applied to the identification of 5 Panax species (P. ginseng, P. japonicus, P. quinquefolius, P. notoginseng and P. vietnamensis). A set of specific primers, including 2-pair primers on chloroplast trnK gene and nuclear 18S rRNA gene regions, respectively, was designed and synthesized for each species on the basis of species-specific sequences of the 2 genes. By using 5 sets of specific primers, in turn, PCR amplifications were performed with total DNA extracted from 5 Panax species as template under appropriate condition, and each resulting product was detected by agarose gel electrophoresis. The results showed that two expected fragments, one from trnK gene and another from 18S rRNA gene regions, were observed simultaneously only when the set of species-specific primers encountered template DNA of the corresponding species. This assay could give more reliable results for identification of not only 5 Panax species but also corresponding Ginseng drugs by simultaneous detection of 4-site nucleotide differences on 2 completely different genes.

References

• 1 Shaw P C, But P P. Authentication of Panax species and their adulterants by random-primed polymerase chain reaction.  Planta Med. 1995;  61 466-9
• 2 Cheung K S, Kwan H S, But P P, Shaw P C. Pharmacognostical identification of American and Oriental ginseng roots by genomic fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR).  J Ethnopharmacol. 1994;  42 67-9
• 3 Wang J, Ha W Y, Ngan F N, But P P, Shaw P C. Application of sequence characterized amplified region (SCAR) analysis to authenticate Panax species and their adulterants.  Planta Med. 2001;  67 781-3
• 4 Ha W Y, Shaw P C, Liu J, Yau F C, Wang J. Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD).  J Agric Food Chem. 2002;  50 1871-5
• 5 Zhu S, Fushimi H, Cai S Q, Komatsu K. Phylogenetic relationship in genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences.  Planta Med. 2003;  69 647-53
• 6 Fortina P, Dotti G, Conant R, Monokian G, Parrella T, Hitchcock W, Rappaport E, Schwartz E, Surrey S. Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification refractory mutation system (MARMS).  PCR Methods Appl. 1992;  2 63-6
• 7 Ferrie R M, Schwartz M J, Robertson N H, Vaudin S, Super M, Malone G, Little S. Development, multiplexing, and application of ARMS test for common mutation in the CFTR gene.  Am J Hum Genet. 1992;  51 251-62
• 8 Fushimi H, Komatsu K, Isobe M, Namba T. Application of PCR-RFLP and MASA analyses on 18S ribosomal RNA gene sequence for the identification of three Ginseng drugs.  Biol Pharm Bull. 1997;  20 765-9
• 9 Komatsu K, Zhu S, Fushimi H, Qui T K, Cai S Q, Kadota S. Phylogenetic analysis based on 18S rRNA gene and matK gene sequences of Panax vietnamensis and five related species.  Planta Med. 2001;  67 461-5