中国中药杂志

2017, v.42(13) 2467-2472

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快速PCR方法在哈蟆油真伪鉴别中的应用研究
Application of rapid PCR to authenticate Ranae Oviductus

何志一;唐先明;刘建辉;袁媛;蒋超;赵玉洋;王洋;
HE Zhi-yi;TANG Xian-ming;LIU Jian-hui;YUAN Yuan;JIANG Chao;ZHAO Yu-yang;WANG Yang;Harbin Food and Drug Inspection Center;State Key Laboratory Breeding Base of Dao-di Herbs,National Resources Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

摘要(Abstract):

该研究依据哈蟆油Cyt b基因155位SNP位点设计位点特异性PCR引物,采用碱裂解法提取基因组DNA,2步循环PCR程序进行PCR反应,并对PCR反应条件进行优化,从而建立起一种哈蟆油及其4种常见混伪品的快速PCR鉴别方法。该方法在90℃变性3 s,62℃退火延伸20 s进行32个循环时,加入2μL 100×SYBR GreenⅠ染料,正品显示出明亮绿色荧光,而混淆品不显示荧光。该方法准确、简便,40 min内即可完成鉴别,为哈蟆油药材现场快速鉴定提供技术支持。
Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism,DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized.The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100 × SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s,62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes. The established method provides the technical support for authentication of the Ranae Oviductus.

关键词(KeyWords): 快速PCR;哈蟆油;分子鉴定;荧光检测
rapid PCR;Ranae Oviductus;molecular authentication

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基金项目(Foundation): 中医药行业科研专项(201407003)

作者(Author): 何志一;唐先明;刘建辉;袁媛;蒋超;赵玉洋;王洋;
HE Zhi-yi;TANG Xian-ming;LIU Jian-hui;YUAN Yuan;JIANG Chao;ZHAO Yu-yang;WANG Yang;Harbin Food and Drug Inspection Center;State Key Laboratory Breeding Base of Dao-di Herbs,National Resources Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

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