中药材三斑海马DNA指纹鉴定方法及检测试剂的研究  

Research on DNA fingerprint identification method and detection reagent of Hippocampus trimaculatus of traditional Chinese Medicine

作　　者：吴楠 孟媛 周童 张宇彤 李洋洋 刘琳 李亚茹 艾金霞 高丽君 母润红 WU Nan;MENG Yuan;ZHOU Tong;ZHANG Yu-tong;LI Yang-yang;LIU Lin;LI Ya-ru;AI Jin-xia;GAO Li-jun;MU Run-hong(School of Medical Technology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院,吉林132013

出　　处：《药物分析杂志》2023年第6期1053-1060,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：吉林省科技发展计划项目资助(20200404152YY);吉林省科技发展计划项目资助(20200403047SF);吉林省科技发展计划项目资助(20210204185YY)。

摘　　要：目的:建立中药材三斑海马DNA指纹鉴定方法,研制三斑海马DNA检测试剂盒。方法:利用生物信息学对海马种属的Cytb基因进行分析,以该基因作为靶基因,设计特异性引物,研制三斑海马DNA提取及PCR快速检测试剂,应用分子克隆及测序技术,克隆三斑海马阳性对照药材,并对快速检测试剂盒的特异性、稳定性、重复性及灵敏度进行考察,对市售三斑海马样品进行真伪鉴定。结果:基于Cytb序列可对三斑海马及其混伪品进行区分。自主研发的三斑海马快速检测试剂提取的海马DNA纯度和浓度均可达到PCR的要求,在退火温度为61.5℃时引物特异性最强;克隆测序后三斑海马DNA序列与海马mtDNA Cytb特异指纹区段序列一致;自主研制的试剂重现性、稳定性良好,检测浓度最低限为10^(-3)ng·μL^(-1);对17个市售样品进行检测,合格率为88.2%。结论:建立了一种特异、准确的检测中药材三斑海马DNA指纹鉴定方法;研制的三斑海马DNA检测试剂盒操作简便,灵敏度高,检测结果稳定。Objective:To establish a DNA fingerprint identification method of Hippocampus trimaculatus of traditional Chinese medicine and develop a DNA detection kit of Hippocampus trimaculatus.Methods:The cytb gene of hippocampal species was analyzed by bioinformatics.And the gene was used as the target gene to develop DNA extraction and PCR rapid detection reagent for Hippocampus trimaculatus.The positive control medicine of Hippocampus trimaculatus was cloned by using molecular cloning and sequencing technology.The specificity,stability,repeatability,and sensitivity of the rapid detection kit were investigated,and the authenticity of Hippocampus trimaculatus samples in the market was identified.Results:Based on cytb sequence,Hippocampus trimaculatus and its adulterants could be distinguished.The kit could detect hippocampal components effectively.Primer was strongest at an annealing temperature of 61.5℃.The DNA sequence of Hippocampus trimaculatus after clonal sequencing was consistent with the sequence of the specific fingerprint segment of hippocampal mtDNA Cytb.The minimum detection concentration was 10^(-3)ng·μL^(-1).The self-developed reagent has good reproducibility and stability,17 commercial samples were tested,and the qualified rate was 88.2%.Conclusion:A specific and accurate DNA fingerprinting method for detecting hippocampal DNA of Hippocampus trimaculatus Chinese medicine has been established.The developed DNA detection kit for Hippocampus trimaculatus has the advantages of simple operation,high sensitivity and stable detection results.

关 键 词：三斑海马 DNA指纹鉴定 CYTB 检测试剂 DNA提取方法 聚合酶链式反应 分子克隆 

分 类 号：R917[医药卫生—药物分析学]