白花蛇舌草的 FINS 方法鑑定   全文下載

Value of FINS Depends on Choice of DNA Locus as Shown in Baihuasheshecao

FINS （Forensically Informative Nucleotide Sequencing）是一種以DNA序列為基礎的分子生物鑑定方法，利用檢體與參考物種間的簇集關係，透過物種親緣關係分析，達到分子鑑定的目的。選擇合適的DNA序列對於FINS鑑定的成功至關重要，本研究評估6種DNA片段（ITS, matK, rbcL, rps16, trnH-psbA 和trnL-trnF）應用FINS方法，鑑定治療癌症的耳草屬白花蛇舌草（Hedyotis diffusa Willdenow）。 36個耳草屬樣品和8個白花蛇舌草來自香港、澳門、臺灣、中國大陸及美國。研究結果，形成單一簇集的比率由高至低分別為ITS（89%），rps16（78%），matK（74%），trnL-trnF（72%）， trnH-psbA（67%）和rbcL（61%）。所有的DNA片段均可以區分白花蛇舌草與常見的偽品傘房花耳草、松葉耳草、纖花耳草。ITS，trnH-psbA和trnL-trnF的鑑定結果，5個白花蛇舌草是正品，3個是源自於傘房花耳草的偽品。matK，rbcL和rps16無法於實驗檢體擴增DNA片段，因此不適用FINS方法。FINS方法鑑定的成功與否取決於所選取的DNA片段是否可以分辨正偽品及其近親物種，以及是否可以產生可靠的DNA序列，最佳之FINS鑑定白花舌蛇草的DNA片段依次為ITS > trnL-trnF > trnH-psbA > rps16 > matK > rbcL。"

Forensically informative nucleotide sequencing (FINS) is a molecular approach to identify biological materials based on DNA sequences. It focuses on the clustering of samples with reference species through an analysis of the phylogenetic relationship among the species. The choice of loci is critical to the successful use of FINS for identification. In this study, we investigated the influence of six loci (ITS, matK, rbcL, rps16, trnH-psbA and trnL-trnF) on the application of FINS for the identification of traditional Chinese medicine as demonstrated in Baihuasheshecao, a traditional anti-tumor herb derived from Hedyotis diffusa willd. A total of 36 plant samples of Hedyotis species and eight samples of Baihuasheshecao commodities were collected from Hong Kong, Macao, Taiwan, mainland China and the USA. Internal transcribed spacers (ITS) sequences displayed the highest rate of formation of single species cluster (89%), followed by rps16 (78%), matK (74%), trnL-trnF (72%), trnH-psbA (67%) and rbcL (61%). All the loci could discriminate H. diffusa from the common substitute species H. corymbosa, H. pinifolia and H. tenelliflora. FINS identification based on ITS, trnH-psbA and trnL-trnF revealed that five Baihuasheshecao commodities were genuine herb derived from H. diffusa, while the other three commodities were substitutes derived from H. corymbosa. MatK, rbcL and rps16 could not be amplified from all commodity samples and were thus inapplicable for FINS identification of dried the samples. In conclusion, the success of FINS identification depends on the DNA loci as they show different abilities to differentiate genuine species from substitutes, to discriminate closely related species and to generate quality sequences. In this study, the eligibility of DNA locus for FINS identification of Baihuasheshecao commodities, in descending order, is ITS > trnL-trnF > trnH-psbA > rps16 > matK > rbcL.