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基于物种特异性PCR和随机扩增多态性DNA构建的线粒体DNA指纹图谱鉴定鳖甲及其特征

Identification and characteristics of Testudinis Carapax et Plustrum based on fingerprint profiles of mitochondrial DNA constructed by species-specific PCR and random amplified polymorphic DNA.

作者信息

Li Mingcheng, Wang Miao, Zhou Yuqing, Li Zitong, Yuan Guangxin, Wang Xuesong, Xia Wei, Chen Jiayu

机构信息

School of Laboratory Medicine, Beihua University, Jilin, People's Republic of China.

School of Pharmacy, Beihua University, Jilin, People's Republic of China.

出版信息

Mitochondrial DNA B Resour. 2018 Oct 31;3(2):1009-1012. doi: 10.1080/23802359.2018.1507631.

Abstract

Traditional use of Testudinis Carapax et Plustrum (TCP) as a medicine and health food has been widely reported. We compared two DNA fingerprint profiles of mitochondrial (mtDNA) from TCP based on species-specific PCR and random amplified polymorphic DNA (RAPD) to identify their authority. A series of sequences from cytochrome b ( b) of and their counterfeits were downloaded from the Genbank, and Premier 5.0 software was used to design a set of primers. A species-specific PCR and RAPD were undertaken to obtain the different DNA fingerprints respectively. The mtDNA was successfully extracted from all samples using the modified salting-out method. A relative molecular mass of 16.6 × 10 bp was observed, and mtDNA was measured between 1.83 ± 0.02. Fragments of 78 bp were amplified from all the TCP samples tested (except adulterant animals) using species-specific PCR method. The RAPD showed different electrocardiogram between genuine and counterfeit tortoise shell goods along with stripe number and location. The salting-out method (as modified) was used to extract high-quality mtDNA from TCP. The species-specific PCR and RAPD assay proposed in this study could be used for quality control of TCP with more specificity, sensitivity, and applicability.

摘要

龟甲作为药物和保健食品的传统用途已有广泛报道。我们基于物种特异性PCR和随机扩增多态性DNA(RAPD)比较了龟甲线粒体(mtDNA)的两种DNA指纹图谱,以鉴定其来源。从Genbank下载了一系列龟及其伪品的细胞色素b(cyt b)序列,并使用Premier 5.0软件设计了一组引物。分别进行物种特异性PCR和RAPD以获得不同的DNA指纹图谱。采用改良盐析法成功从所有样品中提取了mtDNA。观察到相对分子质量为16.6×10 bp,测得的mtDNA在1.83±0.02之间。使用物种特异性PCR方法从所有测试的龟甲样品(除掺假动物外)中扩增出78 bp的片段。RAPD显示正品和假冒龟甲制品之间的电泳图谱在条带数量和位置上存在差异。改良后的盐析法用于从龟甲中提取高质量的mtDNA。本研究提出的物种特异性PCR和RAPD分析方法可用于龟甲的质量控制,具有更高的特异性、灵敏度和适用性。

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