Real-time PCR And SSR Technology In Identification Of Medicinal Dendrobium Lobe

Stems of genus Dendrobium (Orchidaceae) have been traditionally used as herbal medicine "Dendrobii Herba" inChina."Zipi Fengdou" processed by the stems of Dendrobium devonianum Paxt, which is one of the "Dendrobii Herba" product has become big business as restorative food in China. Due to the decreasing wild resources and the increasing demand,"Zipi Fengdou" commodities present the constant risk of adulteration with cheaperproducts made fromother Dendrobium species in the domestic "Fengdou" market. Different Dendrobium species are so similar in morphology that it is insufficient to identify D. devonianumby the morphological characters, especially when the plants do not blossom.In this study, as a complement to the morphological identification,two detection systemsof D. devonianumwere developed based on SYBR Green II real-time PCR and TaqMan Probereal-time PCR, respectively. The specific site used in the two methods both target on the sequence of the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS) which includes ITS1,5.8S rDNA and ITS2. In the detection system based on SYBR Green II real-time PCR, several specific primer pairsweredesigned by the software Primer Premier6and Oligo6.0, and two of them (ITS1-F/ITS1-R, ITS2-F/ITS2-R) which amplified163bp and159bp fragments situated in ITS1and ITS2of D. devonianum separately were verified to be appropriate for the identification. We can discern the significant differencebetween the average cycle threshold (Ct) value of D. devonianum (15.94for ITS1-4F/ITS1-4R,16.38for ITS2-1F/ITS2-1R) and that of the reference Dendrobiumspecies (34.94for ITS1-4F/ITS1-4R,34.67for ITS2-1F/ITS2-1R)(P<0.001,[SPSS]). Moreover, in thedetection system based on TaqMan Probe real-time PCR,2TaqMan probesTaqMan-DdITS1P/(TaqMan-DdITS1F/TaqMan-DdITS1R)、 TaqMan-DdITS2P/(TaqMan-DdITS2F/TaqMan-DdITS2R) were designed and tested for the quantitative detection of "Zipi Fengdou".The results showed thatthis system can specifically, sensitively and efficiently distinguish the fresh materials and "Fengdou" samples of D. devonianum from those of other Dendrobium species.Furthermore, another identification system based on SSR was also established. The genetic differentiation of three Dendrobium species including D. devonianum, D. officinale and D. moniliforme were analyzed according to the universal SSR markers. And these SSR markers can identify three Dendrobium species mentioned above exactly. Therefore, thesethree identification systems would be adopted by the Quality Control Department for the on-site detection of "Fengdou" in the future.