摘要
目的:针对来源于中华大蟾蜍Bufo bufo gargarizans Cantor的蟾酥建立特异性PCR分子鉴定方法。方法:根据中华大蟾蜍及其混淆品线粒体中编码NADH脱氢酶2(MT-ND2)基因序列的变异位点,设计特异性鉴别引物,提取样品总DNA后建立PCR反应体系,分别对影响特异性PCR反应的主要因素如退火温度、循环次数、DNA模板量、引物量、不同聚合酶及用量、不同品牌PCR扩增仪等进行优化筛选,并对该方法进行了可行性验证。结果:当模板DNA质量符合要求时,中华大蟾蜍样品均在291 bp处有一清晰的特异性条带,而其他样品无此条带出现。结论:该研究所建立的特异性PCR方法可以有效地识别出蟾酥的基原动物中华大蟾蜍,其方法准确度高,稳定性和灵敏度良好,为蟾酥药材的物种真实性鉴定和品质评价提供了技术支持。
Objective:To establish a specific PCR method for rapid detection of Buforvis Venenum from Bufo bufo gargarizans.Methods:According to the mutation site of NADH dehydrogenase 2(MT-ND2)gene sequence encoded by mitochondria of Bufo bufo gargarizans and its mixed counterfeits,specific identification primers were designed and the total DNA of samples was extracted,and PCR reaction system was established.The main factors affecting the specific PCR reaction,such as annealing temperature,cycle num-ber,DNA template amount,primer amount,different polymerase and amount,different brands of PCR amplifiers,were optimized and screened respectively,and the feasibility of the method was investigated.Results:When the quality of the template DNA met the require-ments,all the samples of Bufo bufo gargarizans had a clear specific band at 291 bp,but no such band appeared in other samples.Con-clusion:The specific PCR method established in this research can effectively identify the basic animal of Bufo bufo gargarizans.The method has high accuracy,stability and sensitivity,and provides technical support for the species authenticity identification and quality evaluation of Bufonis Venenum.
作者
黄勇
李宏宇
林梦婷
王乙淋
蒙泳成
HUANG Yong;LI Hong-yu;LIN Meng-ting;WANG Yi-lin;MENG Yong-cheng(Guangxi University of Chinese Medicine/Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine/Key Laboratory of Protection and Untilization of Traditional Chinese Medicine and Ethnic Medicine Resources,Education Department of Guangxi Zhuang Autonomous Region,Nanning 530200,China)
出处
《中药材》
北大核心
2024年第12期2991-2995,共5页
Journal of Chinese Medicinal Materials
基金
广西自然科学基金(2020GXNSFAA238017,2023GXNSFDA026065,2019GXNSFAA245084)
广西高校大学生创新创业训练计划项目(S202210600060,S202310600133)。
关键词
蟾酥
中华大蟾蜍
NADH脱氢酶2基因
聚合酶链式反应
分子鉴定
Bufonis Venenum
Bufo bufo gargarizans Cantor
NADH dehydrogenase 2(MT-ND2)gene sequence
Polymerase chain reaction
Molecular identification
作者简介
黄勇(1982-),男,博士,教授,研究方向:药用动物资源和分子鉴定,E-mail:huangykiz@163.com。
