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目的:建立快速、准确的单核苷酸多态性(SNP)测定法鉴别人参、西洋参及2种样本的混合物。方法:根据ITS和5.8s基因上人参、西洋参的SNP位点设计特异性引物进行PCR扩增,每对PCR引物中有1条引物的3′端正好位于SNP位点上,当互补时发生PCR扩增反应,否则无扩增产物;另1条引物决定着PCR产物的长度。用芯片电泳法检测PCR产物的长度,根据PCR的电泳条带大小进行鉴别。为了减少非特异性扩增,在引物3′端的第3个碱基处人为引入1个错配碱基。结果:当退火温度为55℃时,出现252bp条带的为人参,430bp条带的为西洋参。该鉴别反应特异性高、重现性好、操作简便,能在同一反应中准确鉴别人参、西洋参。一系列不同比例的混合样本测定结果表明,可以检出5%的掺杂成分。电泳分离时间小于100s。结论:通过测定人参和西洋参基因组DNA中的SNP,可以进行品种鉴别和质量控制,可用于中药材的快速鉴定。
Objective: To establish a rapid and accurate single nucleotide polymorphism (SNP) assay for the identification of ginseng, American ginseng and a mixture of two samples. Methods: According to the SNP loci of ITS and 5.8s genes, specific primers were designed for PCR amplification. The 3 ’end of each PCR primer was located at the SNP locus. When complementary, PCR Amplification reaction, otherwise no amplification product; the other one primer determines the length of the PCR product. The length of the PCR product was detected by chip electrophoresis and identified according to the size of the electrophoresis band of the PCR. To reduce nonspecific amplification, a mismatch base is introduced at the third base of the 3 ’end of the primer. Results: When the annealing temperature was 55 ℃, 252bp bands appeared as ginseng and 430bp bands as American ginseng. The identification reaction specificity, good reproducibility, easy to operate, can accurately identify the same reaction ginseng, American ginseng. A series of different proportions of the mixed sample measurement results show that 5% of the doping components can be detected. Electrophoresis separation time is less than 100s. Conclusion: The determination of SNP in genomic DNA of Panax ginseng and Panax quinquefolium can identify and control the varieties, which can be used for the rapid identification of Chinese herbal medicines.