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

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..