中药材龟甲基因组DNA提取及PCR鉴定特征

Identification and Characteristics of Carapax et Plustrum Testudinis Based on PCR Technology and Modified Extraction of Genomic DNA

目的探讨中药材龟甲基因组DNA提取方法,建立聚合酶链式反应(PCR)技术鉴别龟甲真伪的分子指纹特征.方法采用改良盐析法提取新鲜龟甲、龟甲易混品种及市售龟甲样品的基因组DNA.从Gen Bank数据库中下载中华金龟、中华草龟及5种常见伪品龟甲细胞色素b的基因序列,应用Premier 5.0引物设计软件设计特异性引物,对龟甲样品进行PCR扩增.结果改良盐析法提取新鲜龟甲样品DNA纯度均在(1.80±0.12)之间,基因组DNA大小为16.6×103bp.所设计的特异性引物只能扩增正品龟甲,扩增片段为78 bp,而伪品龟甲不能扩增出相应片段.结论改良盐析法可以从龟甲样品中提取到较高质量的基因组DNA.PCR技术鉴别中药材龟甲真伪具有特异性高、方法简便快捷、实用性较强的特点,在中药材龟甲真伪鉴别方面具有较高的应用价值.

Objective To explore extracting method of genomic DNA from Carapax et Plustrum Testudinis and create apolymerase chain reaction （PCR）technology for identifying the molecular finger print characteristics of its authenticity. Method A modified salting out method was used to extract genomic DNA from fresh Carapax et Plustrum Testudinis, its counterfeits and it in market. A series of sequences from cytochrome b of Chinemys reevesi and their counterfeits were downloaded from the GenBank, and Premier 5.0 software was used to design a set of primers. PCR technology was performed out to amplify the specific fragment. Results The genomic DNA were successfully extracted from all samples by the modified salt extracting method. A relative molecular mass of 16.6× 103 bp was observed and genomic DNA was measured between 1.83 ±0.12. The designed specific primers were only used to amplify the genuine products and the amplified fragments were 78 bp, while the corresponding fragments could not be amplified in the false products. Conclusion The modified salting out method could be used to extract high quality of genomic DNA from Carapax et Plustrum Testudinis. The PCR assay proposed in this study could be used for the quality control of Carapax et Plustrum Testudinis, with more specificity, sensitivity and applicability.