基于双重PCR技术的鹿茸及其伪品DNA指纹特征和鉴定

doi:10.13481/j.1671-587x.20180428

吉林大学学报(医学版) ›› 2018, Vol. 44 ›› Issue (04): 839-844.doi: 10.13481/j.1671-587x.20180428

基于双重PCR技术的鹿茸及其伪品DNA指纹特征和鉴定

高丽君¹, 何程远¹, 李盈诺², 巴宏宇¹, 李梓僮², 夏薇¹, 李明成¹, 苑广信², 张丽华³, 艾金霞¹

1. 北华大学医学检验学院临床血液与体液检验教研室, 吉林吉林 132013;
2. 北华大学药学院药物分析教研室, 吉林吉林 132013;
3. 吉林雷宁食品药品检测技术服务有限公司, 吉林吉林 132013

收稿日期:2017-10-30 出版日期:2018-07-28 发布日期:2018-07-27
通讯作者: 艾金霞,副教授,硕士研究生导师(Tel:0432-64608115,E-mail:1047085280@qq.com) E-mail:1047085280@qq.com
作者简介:高丽君(1969-),女,吉林省吉林市人,副教授,医学硕士,主要从事中药DNA检验方面的研究。
基金资助:
吉林省科技厅重点科技成果转化项目资助课题（20160307030YY，20170307001YY）；吉林省发改委产业技术研究与开发项目资助课题（2015Y077）；吉林省教育厅"十二五"科学技术研究项目资助课题（吉教科合字[2015]D143）；国家级大学生创新创业训练计划项目资助课题（201711923018）；吉林省科技厅重点科技攻关项目资助课题（20160204004NY）

Characteristics and identification of DNA fingerprint of velvet antler and its counterfeits based on duplex PCR technique

GAO Lijun¹, HE Chengyuan¹, LI Yingnuo², BA Hongyu¹, LI Zitong², XIA Wei¹, LI Mingcheng¹, YUAN Guanxin², ZHANG Lihua³, AI Jinxia¹

1. Department of Clinical Hematology and Fluid Examination, School of Laboratory Medicine, Beihua University, Jilin 132013, China;
2. Department of Pharmaceutical Analysis, School of Pharmacy, Beihua University, Jilin 132013, China;
3. Jilin Leining Food and Drug Testing Services Co. Ltd, Jilin 132013, China

Received:2017-10-30 Online:2018-07-28 Published:2018-07-27

摘要/Abstract

摘要： 目的：分析鹿茸线粒体细胞色素b （Cytb）和细胞色素C氧化酶亚基Ⅰ（COⅠ）基因特异性，建立双重PCR技术鉴别鹿茸真伪的分子指纹特征。方法：利用碱变性法提取梅花鹿茸、马鹿茸、驯鹿茸和新西兰鹿茸的基因组DNA，应用引物设计软件Premier 5.0针对Cytb和COⅠ分别设计特异性引物（分别为Cytb1、2和COⅠ1、2、3），采用单一及双重引物分别进行PCR扩增，筛选特异性强的引物，确定最佳PCR反应条件。结果：采用碱变性法提取的鹿茸基因组DNA片段长度为23 000bp，DNA纯度即A （260）/A （280）为1.80±0.02；应用单一引物进行PCR扩增无法鉴定鹿茸的真伪，而引物Cytb 1和COⅠ1组合后，解链温度为58℃时，梅花鹿茸（吉林、安徽）、马鹿茸均能扩增出395和525bp大小的2个片段，而驯鹿茸和新西兰鹿茸均未能扩增出相应片段；采用该提取方法及最优化的PCR反应条件，对市售样品进行检测，检测结果与实际情况完全一致。结论：双重PCR技术可从分子水平鉴别鹿茸的真伪，该方法特异性高、实用性强，且简便快捷，在鹿茸真伪鉴别方面具有较高的应用价值。

关键词: 细胞色素C氧化酶亚基Ⅰ, 鹿茸, 双重聚合酶链反应, 细胞色素b, DNA指纹

Abstract: Objective:To analyze the gene specificities of mitochondrial cytochrome b (Cytb) of velvet antler and cytochrome C oxidase subunit Ⅰ (COⅠ),and to establish the duplex polymerase chain reaction(PCR)technique for identifying the molecular fingerprint characteristics of velvet antler. Methods:A modified alkaline method was used to extract the genomic DNA from the pilose antler of Cervus Nippon Temminck,Cervus elaphus Linnaeus,Rangifer tarandus and New Zealand deer.Based on Cyt b and COⅠ genes,the specific primers were designed with Premier 5.0 software(Cytb1,2 and CO Ⅰ 1,2,3);PCR amplification was carried out by the single and duplex primers,and the best PCR conditions and highly specific primers were determined. Results:The length of genomic DNA fragment extracted by the modified alkaline method was 23 000bp,and the DNA purity was 1.80±0.02;PCR amplification by the single primer did not identify the authenticity of velvet antler;when Cytb1 and COⅠ1 were applied to PCR, the annealing temperature was 58℃,the fragments of 395 bp and 525 bp were specifically amplified from both Cervus Nippon Temminck and Cervus elaphus Linnaeus antler(Jilin,Anhui),but no fragment appeared for the Rangifer tarandus and New Zealand pilose antler. By using the determined extraction method and the optimal PCR condition,the commercial velvet antlers were carried out and the test results were identical with the reality. Conclusion:The duplex PCR technique can distinguish the authenticity of velvet antler from the molecular level.The method is good in specificity,practicability,and simplicity,and has high application value in the identification of velvet antler.

Key words: velvet antler, cytochrome C oxidase subunit Ⅰ, DNA fingerprint, cytochrome b gene, duplex polymerase chain reaction

中图分类号:

R282.5

高丽君, 何程远, 李盈诺, 巴宏宇, 李梓僮, 夏薇, 李明成, 苑广信, 张丽华, 艾金霞. 基于双重PCR技术的鹿茸及其伪品DNA指纹特征和鉴定[J]. 吉林大学学报(医学版), 2018, 44(04): 839-844.

GAO Lijun, HE Chengyuan, LI Yingnuo, BA Hongyu, LI Zitong, XIA Wei, LI Mingcheng, YUAN Guanxin, ZHANG Lihua, AI Jinxia. Characteristics and identification of DNA fingerprint of velvet antler and its counterfeits based on duplex PCR technique[J]. Journal of Jilin University Medicine Edition, 2018, 44(04): 839-844.

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基于双重PCR技术的鹿茸及其伪品DNA指纹特征和鉴定

Characteristics and identification of DNA fingerprint of velvet antler and its counterfeits based on duplex PCR technique

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