利用DNA条形码检验中药制剂中的羚羊角药材

目的 研究以线粒体细胞色素C氧化酶亚基I(COI)基因作为DNA条形码检验中药制剂中羚羊角的可行性.方法 以不同厂家生产的8种含羚羊角中药制剂为材料,经充分研磨后,采用DNA提取试剂盒提取样品中DNA,以引物LCO1490和HCO2198或特异性引物0703扩增线粒体COI基因片段序列,PCR产物直接双向测序,将测序结果在GenBank中进行BLAST比对,并以DNAMAN和MEGA5.2软件构建同源树和NJ树,以赛加羚羊(gb|JN632700.1)和羚羊角标准药材序列为对照,判定制剂中羚羊角的真实性,并以Kimura-2-parameter模型计算种内、种间遗传距离.结果 8种中药制剂中羚羊角的COI基因序列长度均为658 bp,在同源树和NJ树中可与赛加羚羊和羚羊角标准药材聚成一支,并与outgroup完全分开.种内遗传距离为0～0.064,平均遗传距离为0.020,种间遗传距离为0.167～0.195.结论 以线粒体COI基因作为DNA条形码检验中药制剂中羚羊角方法是可行的,但因羚羊其他器官也含有相同基因,为保证检验结果的准确性,还需配合使用其他检验方法.

Identification of Saigae Tataricae Cornu in Chinese materia medica preparation using DNA barcoding

Objective To discuss the feasibility of using partial sequence of mitochondrial cytochrome C oxidase subunit I (COI) gene as DNA barcoding to identify Saigae Tataricae Cornu (STC) in Chinese materia medica (CMM) preparation. Methods The DNA barcoding identification was constructed in STC samples from eight different manufactories. The genome DNA was extracted by DNA extraction kit from adequately ground samples. The COI nucleotide sequences were PCR amplified with the Primers LCO1490 and HC02198 or specified primer 0703 and sequenced bi-directionally. The BLAST comparison was made in GeneBank, and the phylogenetic trees were constructed with the homologisation tree (DNAMAN) and Neighbor-Joining (NJ, MEGA 5.2) method. Saiga tatarica (gb|JN632700.1) and STC were selected as reference medicinal materials and the genetic distance was calculated using Kimura-2-parameter. Results The lengths of partial mitochondrial COI gene collected from STC in eight samples of CMM preparation were about 658 bp. The phylogenetic tree showed that the samples of CMM preparation and reference substance assembled distinctly, and were completely separated from the outgroup. The intraspecific genetic distances of these samples ranged in 0-0.064 with an average of 0.020, and the interspecific genetic distances ranged in 0.167-0.195. Conclusion The mitochondrial COI gene is a valid DNA barcoding gene for STC identification in CMM preparation. But other organs in antelope also contain the same gene. In order to ensure the accuracy of the results, other texst methods also need to be used cooperatively.