基于DNA条形码鉴定豆蔻类中药材

Identification of Amomi Fructus Rotundus Based on DNA Barcoding

目的:利用ITS2序列对豆蔻类中药材进行DNA条形码鉴定研究。方法:本文采集了包括豆蔻两个基原白豆蔻Amomum kravanh与爪哇白豆蔻Amomum compactum、红豆蔻Alpinia galanga、草豆蔻Alpinia katsumadai等在内的70份样品,采用试剂盒法提取基因组DNA,应用Primer premier 5.0设计引物,通过聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增其ITS2序列,并对PCR产物进行双向测序,所得序列经Codon Code Aligner V3.71拼接后,采用MEGA5.0软件进行序列比对,计算种内和种间距离,构建邻接树(Neighbor-joining tree,NJ Tree)。ITS2序列采用基于隐马尔可夫模型(Hidden Markov model,HMM)的注释方法获得。结果:通过新设计引物扩增的序列可以注释到5.8 S与28 S,扩增序列位于ITS2间隔区。采用新设计的引物,所有实验样品均可获得ITS2序列,NJ树结果显示不同种豆蔻类药材分别聚为一支,可以互相区分,且可以与其混伪品相互区分。另外,通过序列比对发现有5个SNP(s)存在于豆蔻与其它物种种间,且有2个稳定的SNP(s)存在于白豆蔻和爪哇白豆蔻种间,可用于二者的准确鉴定。结论:ITS2序列作为DNA条形码可准确稳定地鉴别不同种豆蔻类药材,本研究将为保障临床安全用药提供新的技术手段。

Objective： In this study, the internal transcribed spacer 2 of nuclear ribosomal DNA （ITS2） sequence was used for identifying the traditional Chinese medicine from Amomum and Alpinia to ensure the quality medicines and clinical efficacy. Genomic DNA was extracted from 70 samples using Genomic DNA kit and used as templates for Polymerase Chain Reaction （PCR）. Sequences assembly and consensus sequence generation were performed by Codoncode Aligner. The intraspecific and interspecific genetic distances were computed and the neighbor-joining tree was constructed by MEGA 5.0 in accordance with the kimura 2-parameter （K-2P） model. The ITS2 regions were obtained by using the hidden Markov model（ HMM ）-based annotation methods from the ITS2 sequence. Results： With the specific designed primers, ITS2 sequences can be obtained from all samples. Every species clustered into a clade in the neighbor-joining phylogenetic trees （NJ tree ）respectively, and can be accurately separated with other varieties and adulterants. In addition, we can find some SNP（s） among Fructus Amomi Rotundus and other experiment species by comparing ITS2 sequences, and Amomum compactum and Amomum kravanh can be distinguished well through two stable SNPs. Conclusion： ITS2 region could be used as an efficient barcoder to identify the traditional Chinese medicine from Amomum and Alpinia. This study could provide a new technique to ensure clinical safety in utilization Chinese medicines.