乳鹿分子身份证快速鉴定方法的建立及检测试剂盒的研制  

Establishment of Fast Identification Method for Lactic Cervi Molecule ID and Development of Detection Kit

作　　者：王艳双[1,4] 姜海瀛 张志杰 孙丽媛 艾金霞 高丽君 李明成 苑广信[3] 张丽华[5] WANG Yan-shuang;JIANG Hai-ying;ZHANG Zhi-jie;SUN Li-yuan;AI Jin-xi;GAO Li-jun;LI Ming-cheng;YUAN Guang-xin;ZHANG Li-hua(School of Medical Faculty,Beihua University,Jilin 132013,China;School of Laboratory Medicine,Beihua University,Jilin 132013,China;School of Pharmacy,Beihua University,Jilin 132013,China;Chinese Medicine DNA Fingerprint Detection Technology Technol ogy Innovation Center,Beihua University,Jilin 132013,China;Jilin Leining Food and Drug Testing Technology Service Co.,Ltd.,Jilin 132013,China)

机构地区：[1]北华大学基础医学院,吉林吉林132013 [2]北华大学医学技术学院,吉林吉林132013 [3]北华大学药学院,吉林吉林132013 [4]北华大学吉林省中药DNA指纹检测技术科技创新中心,吉林吉林132013 [5]吉林雷博科技有限公司,吉林吉林132013

出　　处：《中国药学杂志》2020年第21期1787-1794,共8页Chinese Pharmaceutical Journal

基　　金：吉林省发改委项目资助(2018C048-2);吉林省科技发展计划项目资助(20180201023YY,20190303093SF,20190301014NY,20200404152YY,20200403047SF);吉林省北华大学大学生创新创业训练计划项目资助(202010201191);吉林省科技创新中心建设项目资助(20190902018TC);吉林省食品药品监督管理局项目资助(201708)。

摘　　要：目的建立乳鹿分子身份证快速鉴定方法,研发出乳鹿分子身份证快速检测试剂盒。方法以乳鹿mtDNA Cytb作为靶基因,设计乳鹿特异性引物(扩增片段长度为46 bp),确定乳鹿分子身份证;应用分子克隆及测序技术,克隆乳鹿DNA检测的标准品并验证分子身份证序列的正确性;开发出乳鹿分子身份证快速检测试剂盒,并对试剂的特异性、重现性、稳定性及灵敏度进行考察。结果利用乳鹿mtDNACytb基因设计小片段引物,56℃时特异性最强,乳鹿对照药材及乳鹿正品均在46bp处出现一条特异性扩增条带,而阴性及空白对照均未出现扩增条带,确定了乳鹿的分子身份证;克隆测序后的乳鹿DNA序列与乳鹿mtDNA特异指纹区段序列同源性100%,同时验证了乳鹿分子身份证序列的正确性。自主研发的试剂特异性、重现性、稳定性良好,模板DNA最低检出量为5 pg·μL^-1。结论本实验建立的乳鹿分子身份证快速鉴定方法特异、准确、可靠,研制的试剂盒操作简便、结果稳定,适用于DNA降解严重的材料。OBJECTIVE To establish a fast identification method of Lactic Cervi molecule ID and develop a fast detection kit for Lactic Cervi molecule ID. METHODS With the Lactic Cervi mtDNA Cytb as the target gene, the Lactic Cervi specific primer was designed with an amplified fragment of 46 bp in length, and the Lactic Cervi molecule ID was determined. Molecular cloning and sequencing technology were used to clone Lactic Cervi DNA detection standards and verify the correctness of the sequence of the molecular ID. Then, the fast detection kit of Lactic Cervi molecule ID was developed, and the specificity, reproducibility, stability and sensitivity of the reagent were investigated. RESULTS The designed small fragment primers by using mtDNA Cytb gene of Lactic Cervi showed the strongest specificity at 56 ℃. A specific amplification band appeared at 46 bp for both the control drug and the authentic product of Lactic Cervi, while no amplification band appeared in the negative and blank control, which confirmed the molecule ID of Lactic Cervi. After cloning, the DNA sequence of Lactic Cervi was 100% homologous with mtDNA specific fingerprint segment sequence of Lactic Cervi, which verified the correctness of the sequence of Lactic Cervi molecular ID. The self-developed reagent had good specificity, reproducibility and stability, and the minimum detection amount of template DNA was 5 pg·μL-1. CONCLUSION The fast identification method for Lactic Cervi molecule ID established in this study is specific, accurate and reliable. The detection kit is easy to operate and stable, which is suitable for materials with serious DNA degradation.

关 键 词：乳鹿 分子身份证技术 快速检测试剂盒 分子克隆 测序技术. 

分 类 号：R282.5[医药卫生—中药学]