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Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene
Electronic Journal of Biotechnology ( IF 2.7 ) Pub Date : 2021-01-01 , DOI: 10.1016/j.ejbt.2020.10.005
Yingnuo Li , Yanshuang Wang , Mingcheng Li , Lihua Zhang , Guang Xin Yuan

Abstract Background Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. Results This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. Conclusions The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

中文翻译:

基于线粒体细胞色素 b 基因鉴定尖齿蝽蝾螈的物种特异性 PCR 检测方法的开发

摘要 背景蝮蛇是一种中药,临床用于治疗风湿病、肿瘤、心脑血管疾病。由于尖齿虎的独特药用价值和人工繁育难度大,市场上尖齿虎供大于求,市场上发现了大量其掺假品。本研究对尖齿蛉和9条蛇的cytb基因序列进行比对分析,设计特异性引物,建立特异性PCR方法对市场上尖齿尖齿虎药材进行检测。结果该方法成功地将蛇与其他掺假蛇种区分开来,并对从6个城市随机采购的18份尖齿蝮蛇样本进行了检测。十二个样品是假冒的,六个是真品。Agkistrodon acutus的标准参考物质通过分子克隆和测序进行克隆,与其他物种的基因序列差异显着。表明该区域可以作为目标物种的指纹区域。结论 该方法可作为种特异性标志物,与其他掺假蛇种有较高的区分度,有助于有效避免蝮蛇假售问题。
更新日期:2021-01-01
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