Hao Li, Shi Xin, Wen Shiyu, Yang Caiye, Chen Yaqi, Yue Samo, Chen Jiaqiang, Luo Kexin, Liu Bingliang, Sun Yanxia, Zhang Yi
College of Food and Biological Engineering, Chengdu University, Chengdu, China.
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China.
Front Plant Sci. 2025 Jan 16;15:1492083. doi: 10.3389/fpls.2024.1492083. eCollection 2024.
(Hook.f. & Thomson) H.Ohba, a member of the Crassulaceae family, is a traditional Chinese medicine recognized as the original source of Rhodiolae Crenulatae Radix et Rhizoma in the 2020 edition of the China Pharmacopoeia. It has been widely used in both Asia and Europe to enhance stress resistance and reduce fatigue. However, the classification of species can lead to confusion, raising safety concerns in the herbal medicine market.
The cleaved amplified polymorphic sequence (CAPS) RT-PCR was used to identify the single nucleotide polymorphism (SNP) sites, based on which the loop-mediated isothermal amplification (LAMP) was employed to develop the method in identification. The specific loop backward primers, reaction temperature, reaction time, and color indicators were screened and optimized.
Single nucleotide polymorphism (SNP) sites were identified between Rh. crenulata and two closely related species. Based on the identified SNP sites, the optimal real-time fluorescence LAMP system to identify was constructed with the most efficient specific loop backward primers, reaction temperature. The final detection system exhibited a sensitivity of up to 1,000 copies of the target DNA, maintaining a constant reaction temperature of 62°C within 35 minutes. To facilitate visualization, we incorporated two color indicators, hydroxy naphthol blue (HNB) and neutral red (N-red), into the reaction system.
Collectively, we developed a simple, rapid, specific, sensitive, and visible method to distinguish from other two Rhodiola species and hybrids. This approach has significant potential for applications in pharmaceutical industry.
（胡克福和汤姆森）H.Ohba属于景天科，是一种传统中药，在2020年版《中国药典》中被认定为红景天根茎的原始来源。它在亚洲和欧洲都被广泛用于增强抗应激能力和减轻疲劳。然而，物种分类可能会导致混淆，引发草药市场的安全担忧。
采用酶切扩增多态性序列（CAPS）RT-PCR鉴定单核苷酸多态性（SNP）位点，并在此基础上运用环介导等温扩增（LAMP）技术开发鉴定方法。对特异性环反向引物、反应温度、反应时间和颜色指示剂进行筛选和优化。
在红景天与两个近缘物种之间鉴定出单核苷酸多态性（SNP）位点。基于鉴定出的SNP位点，构建了最佳实时荧光LAMP系统，采用最有效的特异性环反向引物和反应温度进行鉴定。最终检测系统对目标DNA的灵敏度高达1000拷贝，在35分钟内保持62°C的恒定反应温度。为便于观察，我们在反应体系中加入了两种颜色指示剂，羟基萘酚蓝（HNB）和中性红（N-红）。
总体而言，我们开发了一种简单、快速、特异、灵敏且可视化的方法，用于区分红景天与其他两种红景天属物种及其杂交种。该方法在制药行业具有重要的应用潜力。
