利用巢式PCR與DNA定序方法鑑定龜鹿二仙膠中龜板與鹿角   全文下載

Identification of Testudinis Carapax and Cervi Cornu in Kuei-Lu-Erh-Hsien-Chiao by Nested PCR and DNA Sequencing Methods

龜鹿二仙膠為本地民間常用之中藥製劑，常有民眾非法製造販售情形。龜板與鹿角是龜鹿二仙膠的主要藥材成分。本研究即應用巢式PCR 與DNA 定序，建立龜鹿二仙膠中龜板與鹿角基原的鑑定方法。研究中所使用的DNA 標記為12S rRNA 基因序列，以此作為鑑別依據，同時修正DNA萃取方法，以確保能萃取到檢體中微量的DNA。依據標準品及GenBank之DNA 序列資料，分別設計能專一擴增鹿及龜DNA 片段的兩對引子，對檢體進行巢式PCR 及DNA 定序。結果顯示3 件合法廠商製造之龜鹿二仙膠，均能檢出龜及鹿的DNA，而26 件地方衛生單位送驗檢體中，僅7 件同時含有龜鹿成分， 16 件含有龜或鹿單一成分，以及3 件為未檢出龜鹿成分之偽品。而本研究之方法確認可應用於鑑定龜鹿二仙膠中龜鹿藥材成分。

Kuei-Lu-Erh-Hsien-Chiao (KLEHC) is widely used in Chinese medicine preparation for folk custom regimen, and is often sold through illegal suppliers. Testudinis Carapax and Cervi Cornu are the main raw materials of KLEHC. This study applied nested PCR and DNA sequencing method to identify the components of turtle and deer ingredients, which are the main constituents of KLEHC. The identification was based on the 12S rRNA gene sequences of mitochondria. This study modified the DNA extraction process to obtain the trace of DNA in KLEHC. With the sequences of the standards and GenBank database, two sets of primers were designed to specifically identify the turtle and deer components respectively. The results showed that all three samples from registered Chinese pharmaceutical manufacturers contained both turtle and deer components. Only seven in 26 samples from unlicensed suppliers contained both turtle and deer components, while 16 samples contained turtle or deer and three did not contain any. The results indicated that the proposed method could be employed to identify turtle and deer components in KLEHC.