目的 本实验对名贵中药材鹿茸的DNA指纹特征进行研究,从分子水平鉴定及分析鹿茸,研发鹿茸DNA检测试剂盒,并对其进行性能评价。方法 以鹿茸细胞色素C氧化酶亚基Ⅰ(CoⅠ)和鹿茸线粒体细胞色素b(cytochrome b, Cyt b)为靶基因,采用生物信息学技术设计5对特异性引物,经实验筛选后确定细胞色素C氧化酶亚基Ⅰ 1,即mt DNA CoⅠ1(NC_013834.1)为特异性最好的引物,对鹿茸样品通过聚合酶链式反应(polymerase chain reaction,PCR)进行扩增,优化PCR反应体系及反应条件,研发出鹿茸DNA检测试剂盒。对其性能进行评价,并对市售鹿茸样品进行检测。结果 使用鹿茸DNA检测试剂盒提取的DNA纯度均在1.785~1.906内,该试剂盒的特异性体现在检测正品时出现条带,而伪品无条带;灵敏度达3.125 ng·μL-1;反复冻融5、10、15、20次后仍有效用;重复检测正、伪品3次,结果均一致;-20 ℃保存,有效期可长达1年。对5个地区12个市售样品进行质量分析,合格率为66.67%。结论 研发的鹿茸DNA检测试剂盒特异性强、灵敏度高、稳定性及重复性均良好,操作简单,检测样品微量,适用范围广,成本低廉,结果准确,可为中药鹿茸提供一种科学、准确、可靠的鉴定方法。同时也表明市售鹿茸样品质量良莠不齐,混伪品大量充斥。
Abstract
OBJECTIVE To study the DNA fingerprint characteristics of the precious Chinese medicinal antler, identify and analyze the velvet antler at the molecular level, and develop the velvet DNA detection kit and evaluate its performance. METHODS Using antler cytochrome C oxidase subunit Ⅰ (Co Ⅰ) and velvet mitochondrial cytochrome b (cytochrome b, Cyt b) as target genes, five pairs of specific primers were designed by bioinformatics technology, and the oxidase subunit Ⅰ of cytochrome C, mt DNA CoⅠ 1 (NC_013834.1), was determined to be the best specific primer by experimental screening. Polymerase chain reaction (PCR) amplification was conducted on velvet antler samples, and the PCR reaction system and reaction conditions were optimized to develop a velvet DNA detection kit. The performance of the kit was evaluated and commercially available antler samples were tested. RESULTS The purity of the DNA extracted by the antler DNA test kit was 1.785-1.906. The specificity of the kit was reflected in the detection of genuine bands, and there was no band in the fakes. The sensitivity was up to 3.125 ng·μL-1. The kit was still effective after 5, 10, 15 and 20 freeze-thaw cycles. The results of repeated detection of positive and negative products for three times were consistent. The kit could be stored for up to one year at -20 ℃. The quality analysis was carried out on 12 commercial samples in five regions, and the pass rate was 66.67%. CONCLUSION The velvet DNA detection kit developed by our team has strong specificity, high sensitivity, good stability and repeatability, with simple operation, small sample volume, wide application range, low cost and accurate results. It provides a scientific, accurate and reliable identification method for Chinese medicinal materials velvet products. At the same time, it also shows that the quality of commercially available velvet samples is uneven, and there are many adulterants.
关键词
分子生物学 /
鹿茸 /
试剂盒 /
评价
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Key words
molecular biology /
antler /
kit /
evaluation
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中图分类号:
R284
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参考文献
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脚注
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基金
吉林省科技厅重点科技成果转化项目资助(20170307001YY);吉林省科技厅技术攻关项目资助(20190301014NY,20190303093SF);吉林省发改委产业技术开发项目资助(2018C048-2);吉林省教育厅“十三五”科学技术项目资助(JJKH20180540KJ)
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