分泌物来源的蟾酥基原物种的分子鉴定

Molecular identification of animal species of Bufonis Venenum from secretions

物种基原是影响蟾酥药材质量的重要因素之一,来源于中华大蟾蜍的蟾酥质量显著高于来源于黑眶蟾蜍的蟾酥。蟾酥属于分泌物,缺乏基原动物的外观形态,传统鉴别法难以鉴别,而分子鉴定技术的快速发展为蟾酥鉴定提供了新的方法。然而,分泌物中残留的DNA含量很少,降解严重,目前对分泌物来源的药材进行分子鉴定研究仍缺乏。为了解蟾酥药材的基原物种,该研究首先收集了83份蟾酥样品,其中71份原动物样品,按照2020年版《中国药典》的方法制备蟾酥,市售蟾酥药材7份,对照药材5份。然后比较了不同DNA提取方法,扩增其线粒体16S rRNA基因片段和构建系统发育树,最后对蟾酥的基原物种进行分子鉴定。结果表明,试剂盒法提取蟾酥DNA质量比较好,83份蟾酥样品中有80份成功扩增出16S rRNA序列。从GenBank上下载了71条蟾酥基原物种的16S rRNA序列。系统发育树结果表明蟾酥基原物种中华大蟾蜍与黑眶蟾蜍各自聚成单系,节点支持率为100%(NJ)和1.00(BI)。市售蟾酥和对照药材的基原物种均为中华大蟾蜍。可见,从分泌物来源的蟾酥能提取DNA,扩增出16S rRNA基因序列,能够对蟾酥基原物种进行分子鉴定,这为蟾酥药材的质量标准、其他分泌物来源的药材基原物种鉴定提供参考。

The animal species is one of the key factors affecting the quality of Bufonis Venenum. The quality of Bufonis Venenum derived from Bufo bufo gargarizans is significantly higher than that from B. melanostictus. Since Bufonis Venenum is from secretions, the conventional identification methods are difficult to identify the animal species due to the lack of the appearance and morphology of the animals. The rapid development of molecular identification technology has provided new methods for the identification of Bufonis Venenum. However, because of the low content and serve degradation of residual DNA in secretions, the research on the molecular identification of Chinese medicinal materials from secretions remains to be carried out. To understand the animal species of Bufonis Venenum, this study collected 83 samples of Bufonis Venenum, including 7 commercially available samples, 5 reference medicinal materials, and 71 animal samples from which Bufonis Venenum was prepared according to the method in the 2020 edition of the Chinese Pharmacopoeia. Different DNA extraction methods were used and compared, and the mitochondrial 16S rRNA gene fragments were amplified, on the basis of which the phylogenetic trees were built. Finally, molecular identification of the animal species of the samples was performed. The results showed that the DNA extracted from Bufonis Venenum by the reagent kit had good quality, and 16S rRNA sequences were successfully amplified from 80 out of the 83 samples. In addition, 71 16S rRNA sequences of the animal species of Bufonis Venenum were downloaded from GenBank. The phylogenetic trees constructed based on the neighbor-joining(NJ) method and the Bayesian inference(BI) method showed that the samples derived from B. bufo gargarizans and B. melanostictus were clustered into separate monophyletic clades, with the support of 100%(NJ) and 1.00(BI), respectively. The animal species of both commercially available samples and reference medicinal materials were B. bufo gargarizans. In conclusion, DNA can be extracted from Bufonis Venenum derived from secretions, and the 16S rRNA gene sequences can be amplified, which can be used for molecular identification of the animal species of Bufonis Venenum. The findings provide a reference for the quality control of Bufonis Venenum and the identification of animal species of medicinal materials derived from secretions.