目的 通过聚合酶链式反应(polymerase chain reaction,PCR)与核酸试纸条相结合的方法,实现鹿茸真伪的可视化检测。方法 采用十二烷基硫酸钠(sodium dodecylsulfate,SDS)碱变性法提取正品及伪品鹿茸样品基因组DNA,通过查找细胞色素C氧化酶亚基I(COI)特异性位点,应用Primer 3.0软件设计鹿茸特异引物,摸索PCR最佳反应体系及条件,建立PCR-核酸试纸条及琼脂糖凝胶电泳鉴定鹿茸真伪的最优条件。同时,通过克隆测序,验证鹿茸真伪鉴定的准确性。结果 本实验中正品鹿茸在PCR-核酸试纸条上均出现两条带,阴性对照及伪品出现一条带,与琼脂糖凝胶电泳结果吻合,且试纸条检测的灵敏度可达1 ng·μL-1。对9个市售鹿茸样品检测,正品鹿茸5个,合格率为45%。结论 自主构建的PCR-核酸试纸条检测方法简单、快速、特异性较好,可在较短时间内实现鹿茸真伪的可视化检测,检测结果稳定,为中药材鉴定提供又一新的方法。
Abstract
OBJECTIVE To investigate deer antler authenticity by combining polymerase chain reaction(PCR) and nucleic acid test strips.CONCLUSION Genomic DNA of genuine and counterfeit antler samples was extracted by alkali denaturation method, and the optimal conditions for the identification of antler authenticity by PCR-nucleic acid test strips and agarose gel electrophoresis were established by searching for cytochrome C oxidase subunit I(COI) specific sites, designing antler-specific primers with Primer 3.0 software, and exploring the optimal PCR reaction system and conditions. Meanwhile, the accuracy of antler authenticity identification was verified by clone sequencing.RESULTS The results of this experiment showed that two bands appeared on the PCR-nucleic acid test strips for genuine antlers and one band for the negative control and pseudo antlers, which were consistent with the results of agarose gel electrophoresis, and the sensitivity of the test strips was up to 1 ng·μL-1. 5 of the 9 commercially available antler samples were tested for genuine antlers, with a pass rate of 45%.CONCLUSION The PCR-nucleic acid strip assay is simple, rapid and specific, and can visualize the authenticity of deer antlers within a short period of time with stable results.
关键词
聚合酶链式反应 /
鹿茸 /
核酸试纸条 /
可视化 /
DNA指纹
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Key words
PCR /
antler antler /
nucleic acid test strip /
visualization /
DNA fingerprinting
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中图分类号：
R282
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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);吉林省科技发展计划项目资助(20200404152YY,20200403047SF);北华大学医学技术学院晨裕科研创新项目资助(202106)
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