利用Nested_PCR-DNA定序方法鑑定中藥製劑中石菖蒲藥材成分

Identification of Acori Graminei Rhizoma in Chinese Medicine Preparations by Nested PCR and DNA Sequencing Methods

石菖蒲藥材基原在中華中藥典記載為Acorus gramineus Soland.之乾燥根莖，而《中華人民共和國藥典》2005版一部記載本品為Acorus tatarinowii Schott.的乾燥根莖，因此本研究是以Acorus gramineus及Acorus tatarinowii的ITS (internal transcribed spacer)序列設計引子，用以鑑定市售石菖蒲中藥製劑中所含石菖蒲成分。檢體以擴增ITS1-5.8S RNA- ITS2序列所設計的引子進行Nested PCR後，無法有DNA片段形成。另重新設計引子，改以僅擴增檢體ITS2片段，經Nested PCR之後，能夠獲得DNA片段產物，將產物定序並分析比對DNA序列，可以獲得鑑定結果。結果顯示，12件市售石菖蒲中藥製劑檢體中，10件檢體檢出石菖蒲成分為Acorus gramineus，1件檢出為Acorus tatarinowii，而有1件未能檢出石菖蒲成分。

As recorded in Pharmacopoeia of Chinese Medicine, Acori Graminei Rhizoma is the dried rhizome of Acorus gramineus Soland. But Acori Graminei Rhizoma is the dried rhizome of A. tatarinowii Schott. in Pharmacopoeia of the People’s Republic of China. In this study, Nested PCR was employed with two specific primer sets based on the DNA sequences of ITS (internal transcribed spacer) of A. gramineus and A. tatarinowii from GenBank, followed by DNA sequencing analysis for the identification of Acori Graminei Rhizoma in sample preparations. DNA of ITS1-5.8S RNA-ITS2 region couldn't be amplified with the primer sets from the extracted total DNA at first. We thus redesigned another primer sets targeted to ITS2 region and obtained the amplicon by nested PCR. The amplicon was sequenced and compared with GenBank database to check which Acori Graminei Rhizoma is in samples. The results showed that ten of twelve samples were identified as Acorus gramineus, one as Acorus tatarinowii and one without contented Acori Graminei Rhizoma.