基于Cyt b基因的PCR技术对牛鞭药材的鉴定方法研究
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篇名: 基于Cyt b基因的PCR技术对牛鞭药材的鉴定方法研究
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摘要: 目的:建立对牛鞭药材的聚合酶链式反应(PCR)鉴定方法,从分子水平上对牛鞭药材的真伪进行鉴定。方法:提取牛鞭及其伪品(驴鞭、猪鞭、羊鞭)的基因组DNA,并对其完整性、纯度和浓度进行检测。利用GenBank相关信息,以牛鞭的线粒体细胞色素b基因(Cyt b)为靶基因,应用Primer-BLAST在线软件设计特异性引物,并对不同物种鞭类样品进行PCR扩增,对产物进行电泳分析;对获得的牛鞭样本PCR产物进行克隆和DNA测序验证。对所建立的PCR鉴定方法进行特异性和重复性考察验证。结果:提取所得的牛鞭及其伪品DNA的纯度较高,无蛋白质或RNA污染;1.5%琼脂糖凝胶电泳结果显示,牛鞭样本在200~300 bp之间均出现明显的目的基因条带,而其他伪品未见相应条带出现。DNA测序结果证实,所获牛鞭样本基因片段的核苷酸序列与GeneBank中牛鞭物种相似性均为100%。方法学验证结果显示所建方法特异性、重复性均良好。结论:本研究所建基于Cyt b基因的PCR鉴定方法简单、快速,准确性、特异性、重复性均良好,可满足牛鞭及其伪品的分析鉴定需求。
ABSTRACT: OBJECTIVE: To establish the method for PCR identification of bullwhip, and to identify the authenticity of bullwhip at the molecular level. METHODS: DNA samples of bullwhip and its counterfeits (donkey whip, pig whip, sheep whip) were extracted and their integrity, purity and concentration were detected. Using GenBank related information, using mitochondrial cytochrome b (Cyt b) gene of bullwhip as target gene, Primer-BLAST online software was used to design specific primer. PCR amplification was performed for whips of different species, and electrophoretic analysis was conducted for the product. PCR products of bullwhip samples were cloned and confirmed by DNA sequencing. The specificity and repeatability of the established PCR method were verified. RESULTS: DNA purity of the bullwhip and its counterfeits was high, and there was no protein or RNA pollution. 1.5% agarose gel electrophoresis showed that there were obvious target gene bands of bullwhip samples at 200-300 bp, while no corresponding bands appeared in other counterfeit products. The results of DNA sequencing showed that the nucleotide sequence of the gene fragment of bullwhip was 100% similar to that of the bullwhip in GeneBank. Results of methodological validation showed that established method was specific and reproducible. CONCLUSIONS: The established PCR identification method based on Cyt b gene in the study is simple, rapid, accurate, specific and reproducible, and can meet the requirements of analysis and identification of bullwhip and its counterfeits.
期刊: 2019年第30卷第24期
作者: 徐岩,段思琪,翟英南,李明成,高丽君,孙丽媛
AUTHORS: XU Yan,DUAN Siqi,ZHAI Yingnan,LI Mingcheng,GAO Lijun,SUN Liyuan
关键字: 牛鞭;鉴定;Cyt b基因;PCR;DNA指纹
KEYWORDS: Bullwhip; Identification; Cyt b gene; PCR; DNA fingerprint
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