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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Gen Bank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。
Objective To study the feasibility of using the mitochondrial cytochrome C oxidase subunit I (COI) gene as a DNA barcode to test the antelope horn in Chinese traditional medicine. Methods Eight kinds of Chinese antelobatrachys herbs prepared by different manufacturers were used as materials. After sufficient grinding, the samples were extracted with DNA extraction kit, and the mitochondrial COI gene fragment was amplified by primers LCO1490, HCO2198 or specific primer 0703. PCR The products were directly bi-directionally sequenced. The sequencing results were BLAST aligned in GenBank, and homologous and NJ trees were constructed by using DNAMAN and MEGA5.2 software. The saiga (gb | JN632700.1) As a control, the authenticity of the antelope horn in the preparation was determined and the intraspecific and interspecific genetic distances were calculated using the Kimura-2-parameter model. Results The length of COI gene in the antelope horn was 658 bp in length for all 8 kinds of traditional Chinese medicine preparations. It could be integrated with the standard saiga and saiga horns in homologous and NJ trees and completely separated from outgroup. The intraspecific genetic distance was 0 ~ 0.064, the average genetic distance was 0.020, and the interspecific genetic distance was 0.167 ~ 0.195. Conclusion Mitochondrial COI gene as a barcode for DNA testing of Antelope horn in Chinese medicine is feasible. However, other organs of Antelopes also contain the same gene. To ensure the accuracy of test results, other test methods should be used in combination.