Study On The Identification Of Dendrobium Officinale Based On Real - Time PCR And ARMS Technology

The famous Traditional Chinese Medicine (TCM) "Tiepi Fengdou" sold in high price is made of the stems of Dendrobium officinale Kimura et Migo. A fast and accurate method for identifying the valuable and endangered TCM ingredients has been established based on the combination of real-time PCR (RT-PCR) and amplification refractory mutation system (ARMS) technology. The method is applied to distinguish true and false materials of fresh, dry, especially the processed D. officinale.Sixty Dendrobium Sw. species were used for primer design, and 25 common species of them which are the common adulterants were selected and more than three samples for each species were tested in our research. The aim of this study is to develop SYBR GreenⅡbased real-time quantitative PCR assay for the authentication of "Tiepi Fengdou". The impact of polysaccharide-high in "Fengdous" on DNA amplification has been reduced to the lowest. The method has successfully authenticated "Tiepi Fengdou" from various "Fengdous" made by the stems of the other Dendrobium species which may be used as the adulterants of "Tiepi Fengdou".A Ct value of about 20.48 was obtained when D. officinale was amplified. However, the fluorescence signal presented significantly higher Ct values (31.83±2.65) for the other species. There is a statistically significant difference between them (P<0.001).This is the first report of the identification of D. officinale using RT-PCR The developed system showed three advantages as follow. First, it overcomes the difficulty that the DNA extracted from processed TCM products has a low quality and yield due to intensive process of plant materials and the high concentrations of polysaccharides in them by using conventional PCR to pre-amplify the internal transcribed spacer (ITS) region of the genomic DNA and used the PCR product as template DNA for the diagnostic RT-PCR. Second, its amplification specificity was significantly increased by introducing mismatched nucleotides into the 3’ends of specific primers according to the ARMS technology. Third, a small 109 bp diagnostic fragment was specifically amplified from the ITS region of D. officinale, but not from that of any reference Dendrobium species. In a word, the system combined ARMS and the real-time PCR for authenticating "Tiepi Fengdou" is sensitive, convenient, and repeatable, and it will play an important role in identifying the genuine "Tiepi Fengdou".