五倍子配方颗粒的位点特异性PCR鉴别  

Dentification of Wubeizi Formula Granules by Allele-specific PCR

作　　者：胡力 陈梓媛 赵玉洋 金艳[2] 张辉 张恬[2] 仝一丹 袁媛[2] 蒋超[2] HU Li;CHEN Zi-yuan;ZHAO Yu-yang;JIN Yan;ZHANG Hui;ZHANG Tian;TONG Yi-dan;YUAN Yuan;JIANG Chao(School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;China Resources Sanjiu Medical&Pharmaceutical Co.,Ltd.,Shenzhen 518029,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]中国中医科学院中药资源中心,北京100700 [3]华润三九现代中药制药有限公司,广东深圳518029

出　　处：《中国现代中药》2023年第8期1668-1675,共8页Modern Chinese Medicine

基　　金：国家自然科学基金项目(82073972);中国中医科学院科技创新工程项目(CI2021A041);科学技术部科技基础资源调查专项(2018FY100800)。

摘　　要：目的:为保障临床用药的安全性和有效性,建立五倍子药材及其加工制品混伪、掺假特异性聚合酶链式反应(PCR)鉴定方法。方法:根据五倍子细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列设计引物,筛选出五倍子的稳定引物区间,并在区间内筛选获得五倍子的特异性单核苷酸多态性(SNP)位点,根据SNP位点设计五倍子特异性鉴别引物WBZ-F、WBZ-R,分别收集五倍子及其混伪品,建立五倍子配方颗粒特异性PCR鉴别方法并优化PCR体系,对该方法进行耐受性和适用性考察。结果:退火温度为53℃,循环36次,五倍子及其配方颗粒经PCR及凝胶电泳后,可在约138 bp处观察到单一明亮的特异性鉴别条带,伪品倍蛋蚜虫虫瘿无条带。结论:建立的特异性PCR鉴别方法可准确鉴别五倍子药材、标准汤剂冻干粉、配方颗粒中的五倍子蚜虫源性成分,也可为其他中药配方颗粒的质量标准研究提供参考。Objective:To ensure the safety and effectiveness of clinical medication by establishing a specific polymerase chain reaction(PCR)method for distinguishing Wubeizi and its processed products from adulterants.Methods:Primers were designed based on the cytochrome C oxidase subunitⅠsequence of Melaphis chinensis(Bell)Baker,and the stable DNA region suitable for the identification of Galla Chinensis was screened out.A pair of specific primers(WBZ-F and WBZ-R)was designed based on the single nucleotide polymorphism site in the stable region.The PCR system was established and optimized,and the tolerance and applicability of the established method was evaluated with Galla Chinensis and adulterant samples.Results:The PCR method with the annealing temperature of 53℃and 36 cycles produced a single band at 138 bp for Galla Chinensis and Wubeizi Formula Granules,while it produced no band for the adulterant Schlechtendalia peitan Tsai et Tang or the negative control.Conclusion:The allele-specific PCR method established in this study can quickly and accurately identify Galla Chinensis in the raw materials and processed products.It provides a reference for research on the quality standards of other Chinese medicinal granules.

关 键 词：五倍子蚜虫 配方颗粒 聚合酶链式反应 

分 类 号：R286[医药卫生—中药学]