基于巢式PCR的紫草鉴别引物筛选  

The screening of identification primers for Arnebiae Radix based on nested PCR

作　　者：刘杰[1] 谷海媛 戴胜云 乔菲[1] 连超杰[1] 郑健[1] 加沙尔·斯哈克 LIU Jie;GU Hai-yuan;DAI Sheng-yun;QIAO Fei;LIAN Chao-jie;ZHENG Jian;JIA Sha-er·SI Ha-ke(National Institutes for Food and Drug Control,Beijing 102629,China;Shenyang Pharmaceutical University,Shenyang 110016,China;Yili Institute of Inspection,Testing and Certification,Yili 835000,China)

机构地区：[1]中国食品药品检定研究院,北京102629 [2]沈阳药科大学,沈阳110016 [3]伊犁哈萨克自治州检验检测认证研究院,伊型835000

出　　处：《药物分析杂志》2024年第5期756-765,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：国家药品监督管理局药品监管科学体系建设重点项目“新技术新方法在中药质量控制中的应用”(RS2024Z006-105);中国食品药品检定研究院学科带头人培养基金(2021X4);新疆维吾尔自治区药品监督管理局智力援疆创新拓展人才计划--“新疆特色民族药研究(以新疆紫草为例)团队”项目。

摘　　要：目的:基于巢式聚合酶链式反应(PCR)理念设计并筛选出可用于高效扩增与鉴别紫草市场样品真伪的特异性引物。方法:针对新疆紫草的ITS序列和紫草非《中华人民共和国药典》品ITS2序列,利用Primer Premier 5软件进行巢式引物设计;比较ITS2通用引物PCR和巢式PCR对紫草药材基因组DNA的扩增效率;基于巢式引物直接扩增紫草基因组DNA,并进行琼脂糖凝胶电泳检测;基于扩增产物片段长度、变异位点覆盖情况对设计的紫草药材特异性引物进行评价。结果:经过Primer Premier 5软件进行引物设计共选出11对引物进行合成;巢式PCR对紫草药材基因组DNA的扩增效率明显优于ITS2通用引物PCR;基于巢式引物直接扩增紫草基因组DNA琼脂糖凝胶电泳检测的结果明显优于ITS2引物直接扩增紫草基因组DNA,且呈单一条带;确定AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/10A、AE-29S/AE-29A共4对引物适用于紫草正伪品鉴定。结论:在DNA条形码鉴定和巢式PCR技术的基础上,确定了4对可以用于有效区分药材市场中主流的新疆紫草和紫草非《中华人民共和国药典》品的特异性引物,为后续紫草药材及其他中药民族药品种的鉴定方法研究与开发提供了可参考的依据。Objective:To design and screen specific primers for efficient amplification and identification of Arnebiae Radix from market based on the concept of nested PCR.Methods:Nested primers was designed using the software of Primer Premier 5 based on the ITS sequence of Arnebia euchroma and the ITS2 sequence of non-pharmacopoeial Arnebiae Radix.The amplification efficiency of genomic DNA by ITS2 universal primers PCR and nested PCR was compared.The genomic DNA of Arnebiae Radix was amplified directly by nested primers and was detected by agarose gel electrophoresis.The specific primers designed for Arnebiae Radix based on the fragment length and variation sites'coverage of the amplified product was evaluated.Results:A total of 11 primers were selected for synthesis after the primers were designed by Primer Premier 5 software.The amplification efficiency of nested PCR was superior to ITS2 universal primers PCR in genomic DNA of Arnebiae Radix.The results of nested primers directly amplified genomic DNA of Arnebiae Radix by agarose gel electrophoresis were better than those of ITS2 primers,and showed a single band.Four pairs of primers,AE-9S/AE-2A,AE-4S/AE-10A,AE-12S/10A,AE-29S/AE-29A,were determined to be suitable for the identification of Arnebiae Radix.Conclusion:On the basis of DNA barcode identification and nested PCR technology,4 pairs of specific primers are identified which can be used to effectively distinguish Arnebia euchroma from the mainstreamed non-pharmacopoeial Arnebiae Radix in the medicinal materials market,providing reference for the subsequent research and development of identification methods for Arnebiae Radix and other traditional Chinese medicines.

关 键 词：紫草 聚合酶链式反应(PCR) 内部转录间隔区2(ITS2) DNA条形码 巢式PCR(nPCR) 

分 类 号：R917[医药卫生—药物分析学]