DNA microarray for authentication of medicinal dendrobium species

Abstract

As a major genus in the Orchidaceae family, Dendrobium covers over 1500 species in worldwide. Fure of them, D. fimbriatum, D. chrysanthum, D. nobile and D. officinale are listed in the latest edition of the Chinese Pharmacopoeia as medicinal material Herba Dendrobii (Shihu) (version 2010). Shihu is of great demand, as according to the Pharmacopoeia it benefits the stomach, promotes the production of body fluid, nourishes yin and eliminates evil-heat, and consequently commands high prices and is frequently adulterated. Many Dendrobium species are over-collected and are now endangered. The aim of this study is to explore the feasibility of using DNA microarray technology as an objective and effective method for the authentication and quality control of Herba Dendrobii. In this work, total DNA was extracted from either fresh or dry plants samples. The internal transcribed spacers (ITS) of rDNA from 28 Dendrobium, two orchid and two non-orchid species were amplified by polymerase chain reaction and their sequences were determined. The ITS1-5.8S-ITS2 sequences and the partial 18S and 28S are about 900 bp in length. The average interspecific sequence difference was 13.14% for the ITS1 and 15.37% for the ITS2. On the other hand, intraspecific difference was much lower, with an average of 1.03% for ITS1 and 1.26% for ITS2. Therefore, sequencing of ITS1 and ITS2 can be used to differentiate the Dendrobium species. The ITS1-5.8S-ITS2 sequences of the 28 Dendrobium, two orchid and two non-orchids species, were spotted on a glass slide and hybridized with either Cy5 or Cy3-labeled ITS2 sequences of individual Dendrobium species. The results indicated that 24 of the 28 Dendrobium species can be differentiated. While the remaining four species were distinguished by their 5S sequences complementarily. DNA microarray for detecting processed medicinal Dendrobium species (Herba Dendrobii) was performed by incorporating the ITS1-5.8S-ITS2 sequences of mixed herbal DNA from two Chinese medicinal formulations on a glass slide. Using fluorescence-labeled ITS2 sequences as probes, the source plants of Herba Dendrobii in two formulations were identified. In conclusion this work shows that DNA microarray is able to differentiate the concerned 28 Dendrobium species, including the four medicinal Dendrobium species listed in the Chinese Pharmacopoeia, and to identify Herba Dendrobii in complex medicinal formulations.