道地药材天麻DNA真伪鉴定试剂盒的研制与评价

Development and Evaluation of DNA Authenticity Identification Kit for Genuine Medicinal Materials Gastrodia Elata

关键词:  天麻  ITS2  DNA提取  真伪鉴定试剂盒  聚合酶链式反应

Key words:  Gastrodia elata  ITS2  DNA extraction  authenticity test kit  polymerase chain reaction

摘要:

目的 建立一种将DNA提取技术与PCR技术相结合的天麻DNA真伪鉴定试剂盒，并对试剂盒性能进行方法学评价。方法 通过美国国家生物技术信息中心(NCBI)查找天麻及其常见伪品紫茉莉根、大丽菊块茎、马铃薯的ITS2序列，应用DNAMAN进行多序列比对，NCBI-Primer-Blast设计天麻特异性引物。改良植物常用DNA提取的CTAB法，确保提取出高效的正品天麻及其常见伪品的基因组DNA，应用紫外分光光度法测其浓度及纯度。优化PCR反应体系，确定试剂盒的组成与反应条件，随机抽样检测市售天麻样品。结果 应用所研制的试剂盒提取样品DNA，纯度OD260 /OD280值为(1.87±0.13)，灵敏度为10 ng·μL^-1，3次重复性检测结果相同，反复冻融 5、10、15、20次对检测效果无影响，于-20 ℃可保存1年，检测10个市售天麻样品，其中7个是正品，3个是伪品。结论 道地药材天麻DNA真伪鉴定试剂盒特异性强，灵敏度高，重复性和稳定性均良好，检测结果准确，适用于天麻及其常见伪品的快速鉴别。

Abstract:

OBJECTIVE To develop a DNA authenticity identification kit of Gastrodia elata that combined DNA extraction technology with PCR technology, and to evaluate the performance of the kit methodologically. METHODS The ITS2 sequences of Gastrodia elata and its common forgeries, such as amabilis root, dahlia tuber and potato, were found by the National Center for Biotechnology Information(NCBI). DNAMAN was used for multi-sequence alignment, and NCBI-primer-blast was used to design specific primers of Gastrodia elata. Improved DNA extraction method to ensure efficient extraction of authentic Gastrodia elata and its common forgeries genomic DNA, UV spectrophotometry was used to measure the concentration and purity. The PCR reaction system was optimized, the composition and reaction conditions of the kit were determined, and the commercially available gastrodia elata samples were randomly sampled. RESULTS The DNA purity OD260/OD280 values of the samples extracted by the developed kit were (1.87±0.13). The minimum detection limit was 10 ng·μL^-1, and the result of repeated detection was the same for 3 times. Repeated freezing and thawing for 5, 10, 15, 20 times had no effect on the detection effect, and it could be stored at -20℃ for 1 year, among 10 commercially available gastrodia elata samples tested, 7 were authentic and 3 were counterfeit. CONCLUSION The DNA authenticity test kit is highly specific, sensitive, reproducible and stable, and the test results are accurate, it is suitable for the rapid identification of asparagus and its common forgeries.