普乐安片DNA条形码分子鉴定研究  

Identification of Pule’an Tablets based on DNA barcoding molecular technology

作　　者：魏妙洁 刘金欣 赵晴 徐晓兰 任萍[5] 李明玥 石林春 孙伟[1] WEI Miao-jie;LIU Jin-xin;ZHAO Qing;XU Xiao-lan;REN Ping;LI Ming-yue;SHI Lin-chun;SUN Wei(Institute of Chinese Materia,China Academy of Chinese Medical Sciences,Beijing 100700,China;Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100193,China;School of Pharmacy,Minzu University of China,Beijing 100081,China;College of Animal Science(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Institute of Agricultural Resources and Regional Planning,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]中国医学科学院、北京协和医学院药用植物研究所,北京100081 [3]中央民族大学药学院,北京100193 [4]福建农林大学动物科学学院(蜂学学院),福建福州350002 [5]中国农业科学院农业资源与农业区划研究所,北京100081

出　　处：《药学学报》2020年第6期1327-1333,共7页Acta Pharmaceutica Sinica

基　　金：北京市自然科学基金(7202136);国家自然科学基金项目(81703659);中国博士后科学基金面上项目(2017M610815)。

摘　　要：建立基于DNA条形码技术的普乐安片处方成分鉴定方法。共收集8个厂家的16份普乐安片样品,分别采用植物和动物基因组DNA提取试剂盒提取样品DNA,所获DNA的ITS2序列扩增成功率均为100%,细胞色素C氧化酶亚基I(COI)序列扩增成功率分别为43.75%和56.25%。对于ITS2序列,12份样品可获得高质量测序峰图,鉴定结果为油菜Brassica campestris,4份样品存在较严重套峰,经克隆测序鉴定为油菜和黑芥B.nigra。对于COI序列通用引物获得的9份PCR产物,其中2份样品可直接进行PCR产物测序,鉴定结果为蚜虫类,7份样品经克隆测序获得82条序列,其中5份样品可检测到意大利蜜蜂Apis mellifera,7份样品均可检测到病原菌或病虫害。为解决COI通用引物因外源污染导致的蜜蜂源检测失败问题,新设计2对蜜蜂属Apis的COI引物,扩增效率均为43.75%,7份PCR产物直接测序的鉴定结果均为意大利蜜蜂A.mellifera。9份未检测到蜜蜂源的普乐安片来自同一厂家生产的3个不同批次,且每个批次含3份样品,推测原料花粉非蜜蜂采集所得。本研究基于ITS2和COI序列建立了普乐安片处方成分的鉴定方法,将为普乐安片的质量控制和市场监管提供科学依据和技术保障。This study aimed to establish a method for identification of the prescription components of Pule’an Tablet based on DNA barcoding technology.Sixteen samples have been collected from 8 different companies,and their DNA were purified using Plant and Animal Genomic Kits.The amplification rates of ITS2 were both 100%,and the amplification rates of COI were 43.75%and 56.25%for these samples’DNA purified using plant and animal kits,respectively.For ITS2,12 samples obtained high-quality sequencing traces and then were identified as containing Brassica campestris.The other 4 samples showed crucial SNP peaks in the sequencing traces,which were assigned to be B.campestris and B.nigra on the basis of cloning and sequencing experiments.For the PCR products of 9 samples from COI universal primers,two samples were directly sequenced and identified as aphids,and 7 other samples were subjected to cloning and sequencing experiments.Finally,we obtained 82 clone sequences and found that Apis mellifera was detected only in 5 of the remaining 7 samples,and pathogens or pests were detected in all these 7 samples.To solve the failure of bee source detection caused by exogenous contaminations based on COI universal primers,we designed two new COI primer pairs of Apis genus.The amplification rates of both primer pairs were 43.75%,and they were identified as A.mellifera.A total of 9 bee source-free Pule’an Tablet samples from 3 different batches were produced by the same company,and each batch contained 3 replicates.Thus,we speculated that raw rape pollen materials for these 9 samples was not collected by bees.This study proposes an identification method for the prescription components of Pule’an Tablets based on ITS2 and COI sequences,which will provide scientific basis and technical guidance for quality control and market regulation of Pule’an Tablets.

关 键 词：普乐安片 油菜花粉 ITS2 COI 鉴定 

分 类 号：R931[医药卫生—生药学]