基于显微鉴别和可视化探针技术的砂仁鉴别研究

Identification of Amomum Villosum Lour. Based on Microscopic Identification and Visual Probe Technology

关键词:  阳春砂  显微鉴别  ITS2  可视化杂交

Key words:  Amomum villosum Lour.  microscopic identification  ITS2  visual hybridization

摘要:

目的 应用显微鉴别和基于ITS2序列差异的可视化探针杂交技术鉴别阳春砂、疣果砂仁、海南假砂仁。方法 采用徒手切片法对砂仁及其混淆品的种子进行制片并观察其显微生物。然后提取阳春砂、疣果砂仁、海南假砂仁的总基因组DNA，PCR扩增其ITS2序列，回收PCR产物并连接T载体，筛选阳性质粒并测序，根据测序所得ITS2序列设计阳春砂的专属荧光探针，并取探针1 ng与砂仁及其混淆品的ITS2 PCR扩增产物进行液相杂交。反应条件：95℃变性5 min，70℃退火30 s，在489 nm荧光下进行激发检测。结果 阳春砂与其混淆品的显微结构区别主要在于色素层的层数和外胚乳细胞的形状。所设计的专属探针仅与阳春砂ITS2 PCR产物特异性结合，而不与其混淆品结合。亦可以通过荧光的强弱来定量鉴别中成药中砂仁的掺伪现象。结论 通过显微鉴别和基于ITS2序列差异的可视化探针杂交技术能够很好地鉴别砂仁的真伪，且不要求中药的具体形式，方法简便快捷、成本低、技术门槛低，开拓了更加便捷、准确的砂仁药材的鉴别方法。

Abstract:

OBJECTIVE To identify Amomum villosum Lour., A. muricarpum Elm., and A. chinense Chun ex T. L. Wu by microscopical identification and visual probe hybridization technology based on ITS2 sequence difference. METHODS The seeds of A. villosum and its adulterants were sliced with bare hands, and the temporary chips were made to observe its microstructure. The total genomic DNA of A. villosum Lour., A. muricarpum Elm. and A. chinense Chun ex T. L. Wu were extracted, the ITS2 sequence was amplified by PCR, the PCR product was recovered and T vector was connected, the positive plasmid was screened and sequenced, the specific fluorescent probe of A. villosum Lour. was designed according to the ITS2 sequence, and 1 ng probe was hybridized with the ITS2 PCR product of A. villosum Lour. and its adulterants. The reaction conditions:denaturation at 95℃ for 5 min, annealing at 70℃ for 30 s, excitation detection was performed at 489 nm. RESULTS The difference of microstructure between A. villosum Lour. and its adulterants lied in the number of pigment layers and the shape of endosperm cells. The special designed probe of A. villosum Lour. was specifically combined with ITS2 PCR products, but not with ITS2 PCR products of A. muricarpum Elm. and A. chinense Chun ex T. L. Wu. The adulteration of A. villosum Lour. in traditional Chinese medicine could also be quantitatively identified by the intensity of fluorescence. CONCLUSION Microscopical identification and visual probe hybridization technology based on ITS2 sequence difference can identify the true and false of A. villosum Lour., and do not require the specific form of traditional Chinese medicine, and the method is simple, fast identification, low cost, and low technical threshold, which opens up a more convenient and accurate identification method of A. villosum Lour..