中藥濃縮製劑中防風藥材之巢式PCR與DNA定序方法鑑定   全文下載

Identification of Saposhinkoviae Radix in Concentrated Chinese Medicine Preparations by Nested PCR and DNA Sequencing Methods

防風藥材為Saposhinkovia divaricata（Turcz.）Schischk.植物的乾燥根部，為中藥方劑及製劑中常用藥材。本研究目的即在發展濃縮製劑中真品防風的聚合?鏈鎖反應（PCR）與DNA定序的分子生物技術鑑定方法。製劑檢體包括17種製劑，檢體DNA的取得是經過含蛋白?的溶解溶液處理、有機溶液萃取以及附有矽質膜（silica membrance）能夠吸附DNA的套組加以純化。四十件純化的濃縮製劑樣品DNA須經過巢式聚合?鏈鎖反應（nested PCR）才能避免干擾，單一地擴增內轉錄區間（internal transcribed spacer,ITS）片段。所有PCR產物經過自動定序儀的定序，並將獲得的序列資料與防風標準品序列及美國國家衛生院的Genbank基因資料庫進行分析比對。結果顯示本研究方法可以克服17種製劑中總計54種藥材的干擾，專一擴增防風藥材的ITS片段供定序確認，40件製劑樣品所含的防風藥材ITS序列均與標準品相同。"

Saposhinkoviae Radix, the dried root of Saposhinkovia divaricata (Turcz.) Schischk., is widely used in Chinese medicine preparations. This paper reports on the development of a nested polymerase chain reaction (nested PCR) and DNA sequencing method able to detect the presence of authentic Saposhikoviae Radix in Chinese medicine preparations. Forty concentrated preparation samples in 17 formulas containing a total of 54 kinds of raw materials were purchased. DNA samples were completely digested with a lysis buffer containing proteinase and sarcosyl, extracted with organic solutions and CTAB, and then purified using a commercial kit (QIAGEN GmbH, Germany) that contained silica membrane to absorb the DNA. The internal transcribed spacer (ITS) regions of 40 preparations were amplified by nested PCR. All PCR products were analyzed by auto-sequencer. We compared resultant sequences with the ITS sequences obtained from the standard and Genbank databases. Saposhinkoviae Radix in all 40 preparations tested were identified as Saposhinkovia divaricata. These results indicate the above is a valid method by which to identify the presence of authentic Saposhinkoviae Radix in Chinese medicine preparations.