基于DNA条形码的翠云草快速鉴定方法研究

目的：基于卷柏属植物DNA条形码鉴定序列，运行TaqMan探针技术探讨翠云草快速鉴定新方法。方法：本文利用DNA条形码鉴定技术对采集的11种32份卷柏属样本提取总DNA扩增ITS2序列，结合Genbank数据库的34种103条序列综合分析，针对翠云草设计特异引物及探针，运用实时荧光PCR检测技术比较序列扩增荧光信号差异实现翠云草的快速鉴定。结果：在所分析的物种中ITS2序列可将翠云草与其它卷柏属近源物种有效区分，TaqMan探针及引物特异性较好，本研究建立的方法在翠云草的鉴别中具有良好的特异性、重复性及灵敏性，甚至可从低至1 mg的样品中提取DNA得到有效检出。结论：基于DNA条形码鉴定序列变异位点信息，运用高特异TaqMan探针实时荧光PCR检测技术，可实现翠云草真伪快速鉴别，具有中药材市场监管应用前景，将为中药材快速鉴定应用研究提供参考。

Rearch on Rapid Identification for Selaginella Uncinata Based on DNA Barcodes

To establish a new method for rapid identification of Selaginella uncinata based on DNA barcoding identification sequence of Selaginella by using TaqMan probe. In the present research, thrity-two samples including eleven species of Selaginella were collected, and the total DNA was extracted and the second internal transcribed spacer (ITS2) sequences were amplified and sequenced using the DNA barcoding identification technology, combining with the published 103 ITS2 sequences from 34 species, the specific primers and TaqMan probe for S. uncinata were designed. The real-time fluorescence quantitative PCR (RT-PCR) was employed to compare the fluorescence signals difference of the sequences amplification to realize the rapid identification of S. uncinata. The results showed that S. uncinata and its near-source species could be successfully distinguished in the analyzed species, the TaqMan probe and primers was favorable. The method established in this study also had good specificity, reproducibility and sensitivity in identification of S. uncinata, it even could effectively detect signals from the DNA samples extracted from as less as 1 mg plant issues. In conclusion, based on the variation sites of DNA barcoding sequences, use of highly specific TaqMan probes with RTPCR, the rapid authenticity identification of S. uncinata could be realized. It would has the prospects of application and supervision of Chinese herb medicine market and provide a reference for rapid identification to study.