Allele-specific primers for diagnostic PCR authentication of Halenia elliptica, a traditional Tibetan medicinal plant

Halenia elliptica D. Don. is a biennial herb belonging to the family Gentianaceae that has a long history of widespread use in traditional Tibetan folk medicine. It has beneficial effects on the liver, and is used to treat gall conditions and various other diseases [1,2]. Based on rDNA ITS sequences of Halenia elliptica and the other samples from Swertia and Lomatogonium, respectively, a pair of allele-specific diagnostic primers, was designed for differentiating H. elliptica from its adulterants (Swertia angustifolia, S. erythrosticta, S. franchetiana, S. punicea, S. macrosperma, S. przewalskii, S. tetraptera and Lomatogonium oreocharis) by PCR. Before the diagnostic PCR, the primer pair, TIS4 and ITS5, for amplifying the whole ITS region was used to validate template DNA and to obtain the appropriate template DNA for the diagnostic PCR. Diagnostic PCRs were performed using the diagnostic primers with the total DNAs of the original plants as a template. When the annealing temperature was raised to 60°C, only the template DNA of H. elliptica could be amplified whereas the diagnostic PCRs of the other samples were all negative. The diagnostic PCRs have been repeated many times and have played an important role in authenticating the herbs of H. elliptica in China. This is a major diagnostic improvement, as all other methods for the specific identification of H. elliptica are more time consuming and practical assay.

Acknowledgements: This research was supported by the Natural Science Foundation of China (NSFC 30770153 to CY Xue) and the Natural Science Foundation of Yunnan (NSFN, 2006C 0050M to CY Xue).

References: [1] Ho, T.N. and Pringle, J.S. (1995) Gentianaceae. In: Wu, Z.Y. and Raven, P.H. eds. Flora of China. Science Press, Beijing and Missouri Botanical Garden, St. Louis, MO., 16:1–139.

[2] Yang YC. Tibetan Medicines. Qinghai People Press, Qinghai. (in Chinese); 1991. p.111.