新型核酸分子等溫擴增技術於中藥材基因體基源鑑定之研究－ 以常見誤用及混用中藥材之基源為例(2-2)

李孟修
中國醫藥大學
中藥品質管制的目的，在於辨別真偽、參雜及品質的優劣，以確保臨床的療效。誤用、混用以及偽劣藥材，對於病情不但沒幫助，甚至危害國人的健康。因此如何有效鑒定及管控中藥材，為當前刻不容緩的重要課題。中草藥分子鑒定( Authentication by molecular biology methods)是運用DNA分子遺傳標記技術(DNA genetic marker technology)，直接分析中草藥原植物的基因型而非表現型(如型態及化學成分)，不受材料來源、環境因素、樣品形態(原品、粉末或片狀)影響，樣品少數即可進行實驗4, 5。具有重複性好、靈敏度高的優點，為中草藥品種鑒定上強大的工具。其他方法如外觀鑒定法、顯微鑒定法，化學分析皆會受藥材的種植環境、採集、儲存和炮製而影響鑒定的結果。由此可知，DNA分子鑒定為中草藥品種鑒定的最佳方法，其準確度在其他方法之上。使用DNA分子鑒定技術的研究方法，是對於中藥材基原的基因多態性在分子層次上的檢測，也是中草藥藥材製造流程中最上游(中草藥品種)的品質管控。恆溫圈環式核酸擴增法(Loop Mediated Isothermal Amplification, LAMP) 7為Notomi等於2000年開發的一種新穎的恒溫核酸擴增方法，其特點是針對目標基因中的6個特異區域設計4種特異引子(Primer)，在恒溫條件下(60—65℃)利用一種具有鏈置換活性(Chain displacement activity)的DNA聚合酶(Bst DNA polymerase)，保溫幾十分鐘，即可完成核酸擴增(Amplification)反應7。並不需要反覆對模版(Template)DNA進行熱變性(Denaturation)及長時間的溫度迴圈。LAMP是一種嶄新的DNA擴增方法，具有簡單、快速、特異性強的特點，具有替代PCR方法的可能性，也為大多源自PCR技術的中草藥分子鑒定技術提供了一種強而有力的新方法。本研究計畫將運用並建立LAMP做為植物基因體鑑別的方法，本計畫在第一年中，即針對中藥材正品與其混用藥材基因組如白花蛇舌草(與多花水線草)、蒲公英(與兔兒菜) 、粉防己(與廣防己)等3種，設計特定引子，並最佳化反應條件、分析方法的檢測靈敏度和特異性，觀察中藥材正品及混用藥材的LAMP檢測效果。而第二年度則選用另外三種模式藥材-十大功勞(與枸骨葉)、白前(與白薇)、及黃耆(與紅耆) 共3種之LAMP快速鑒定方法建立。
關鍵字：中藥品質管制、中藥基原分子鑒定、恆溫核酸擴增法

Study on Genomic Authentication of Chinese Herbal Medicine Using a Novel Isothermal Nucleic Acid Amplification Technology- Creacting an Authentication Model for Mis-identified Medicinal Herb Species and Its Adulterants(2-2)

Meng-Shiou Lee
China Medical University
Quality control plays a vital role in the development of translating the traditional medicines into modern evidence-based therapies. Authentication of traditional Chinese medicine (TCM) is generally recommended for the standardization and quality control of herbal products and herb related investigations. It has been shown that counterfeits and inferior medicinal material reduce TCM quality and cause cases which patients do not recover or even die. Hence, authentication is an essential step for clinical application and accurate experimental studies of TCM. For example, Peh-Hue-Juwa-Chi-Cao (PHJCC) is a common commercial name of H. diffusa in the herbal medicine material market. However, H. corymbosa (HC) has the same common commercial name in the market. The mix material used is not standardized, and the consistency of quality of this herbal is uncertain.In recent years, the DNA molecular biology method have now become a popular method for the identification and authentication of TCM and developed to be a more effective, accurate, reliable and sensitive technology. Loop-mediated isothermal amplification (LAMP) assay is such a potential method that is capable of amplifying DNA with high specificity, rapidity, and efficiency under isothermal conditions. The method is under isothermal conditions using four to six specifically designed primers. One key feature of the LAMP is using Bst DNA polymerase instead of Taq DNA polymerase in the reaction. Therefore, the aim of this study for first yearis to use LAMP assay to rapid authenticate of H. diffusa, T. formosanum, S. tetrandra and its adulterants, . In second years of this study, the LAMP method will be applied for the detection of ITS sequence in three plants and its adulterants such as Mahonia oiwakensis, Cynanchum stauntonii, Astragalus membranaceus. Applying this method, it might be an alternative method to rapid authenticate the TCM at the market in the future.
關鍵字：Quality control, Authentication TCM by molecular biology methods, Isothermal nucleic acid Amplification (LAMP)