School of Basic Courses, Guangdong Pharmaceutial University, Guangzhou 510006, China.
Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Institute of Applied Biological Resources, Guangzhou 510260, China.
Biosci Rep. 2019 Feb 22;39(2). doi: 10.1042/BSR20181943. Print 2019 Feb 28.
Guang-dilong () is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.
杠板归（）是一种传统的中药在中国已经使用了几千年。在本研究中，我们提出建立一种新的杠板归快速鉴定方法。我们提供了一种有用的技术，环介导等温扩增（LAMP），以区分杠板归和其他物种。根据杠板归的线粒体细胞色素氧化酶 I（COI）基因序列，设计了四个特异性的 LAMP 引物。LAMP 反应，包含 DNA 模板、四个引物、10×Bst DNA 聚合酶反应缓冲液、dNTPs、MgSO 和 Bst DNA 聚合酶，在 63°C 下 60 分钟内完成。通过添加 SYBR Green I 可以可视化 LAMP 产物，或通过 2%凝胶电泳进行检测。成功建立了用于快速鉴定杠板归的 LAMP 技术。此外，LAMP 在杠板归中的检测限为 675 fg/μl，是常规 PCR 的 1000 倍。简单、敏感、方便的 LAMP 技术非常适合在草药市场上进行杠板归的现场鉴定。
