金钗石斛的位点特异性PCR鉴别研究

Authentication of Dendrobium nobile by allele-specific diagnostic PCR

目的建立一种简便、实用的金钗石斛的DNA分子鉴别方法。方法根据金钗石斛及其他黄草类、枫斗类石斛的rDNA ITS序列数据库,寻找金钗石斛特异性的序列位点,设计专用于金钗石斛鉴别的PCR引物,用该引物在不同条件下扩增石斛属植物的模板DNA,建立只有金钗石斛具有阳性扩增的PCR鉴别条件。结果在66℃、40 s的复性条件下进行鉴别PCR,金钗石斛的模板DNA扩增出约300 bp的阳性扩增带,而其他39种石斛的模板DNA在同样条件下无扩增产物。结论运用位点特异性PCR能有效地从其他石斛属植物中鉴别出金钗石斛,该方法具有高效、准确、简便、省时的特点,应用前景广泛。

Objective To develop a convenient and effective molecular authentication method for identifying Dendrobium nobile from other species of Dendrobium Sw. Methods Based on rDNA ITS sequences of D. nobile and other species of Dendrobium Sw. of Huangcaos and Fengdous, unusual sequence sites were confirmed and an allele-specific diagnostic primer pair was designed for D. nobile. Diagnostic PCR was performed using the primers with the total DNA of D. nobile and other species of Dendrobium Sw. as templates to establish the positive PCR condition for D. nobile. Results An about 300 bp DNA positive fragment was amplified from D. nobile with anneal temperature at 66℃ and anneal time at 40 s, whereas no any DNA fragment was amplified from the rest 39 species of Dendrobium Sw. Conclusion D. nobile could be effectively distinguished from other species of Dendrobium Sw. by diagnostic PCR with allele-specific diagnostic PCR, which is not only effective and specific, but also simple and timesaving, and has a comprehensive application prospects.