Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal; Laboratório Associado para a Sustentabilidade e Tecnologia em Regiões de Montanha (SusTEC), Instituto Politécnico de Bragança, Bragança, Portugal.
Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal; Laboratório Associado para a Sustentabilidade e Tecnologia em Regiões de Montanha (SusTEC), Instituto Politécnico de Bragança, Bragança, Portugal; LAQV-REQUIMTE & Department of Chemistry, University of Aveiro, Campus Universitário de Santiago, Aveiro, 3810-193, Portugal.
Phytomedicine. 2024 Jun;128:155322. doi: 10.1016/j.phymed.2023.155322. Epub 2023 Dec 30.
Species adulteration is a concern in herbal products, especially when plant substitutes of lower economic value replace valuable botanicals. Styphnolobium japonicum is well known as a potential adulterant of Ginkgo biloba, which is one of the most demanded medicinal plants due to its wide use in pharmaceuticals, food supplements, and traditional medicine. Despite bearing some resemblance to ginkgo's flavonol composition, S. japonicum lacks many of G. biloba's desired therapeutic properties. To prevent adulteration practices, it is crucial to implement rigorous quality control measures, including fast and simple diagnostic tools that can be used on-field.
This study aims to develop for the first time a species-specific loop-mediated isothermal amplification (LAMP) method for the fast identification of S. japonicum in ginkgo-containing products.
A set of four specific primers (SjF3, SjB3, SjFIP, and SjBIP) and loop primers (SjLF and SjLB) were designed for a LAMP based assay using the 5.8S partial sequence and the internal transcribed spacer 2 of nuclear ribosomal DNA of S. japonicum.
The successful amplification of the LAMP assay was inspected through visual detection, with the highest intensity recorded at the optimal conditions set at 68 °C for 40 min. The primers showed high specificity and were able to accurately discriminate S. japonicum from G. biloba and 49 other species of medicinal plants. Furthermore, the proposed LAMP assay proved to be fast, selective, and highly sensitive, as demonstrated by the absolute and relative limits of detection, which were reached at 0.5 pg for S. japonicum DNA and 0.01 % S. japonicum in G. biloba, respectively.
This novel approach allows easy identification and discrimination of S. japonicum as a potential adulterant of G. biloba, thus being a useful tool for quality control. Compared to chromatographic or PCR-based methods, the assay proved to be fast, sensitive and did not require expensive equipment, thus offering the possibly usage in field analysis.
种属掺假是草药产品的一个关注点，特别是当经济价值较低的植物替代品替代有价值的植物时。皂荚是银杏的潜在掺杂物，由于其在制药、食品补充剂和传统医学中的广泛应用，银杏是需求最大的药用植物之一。尽管皂荚在类黄酮组成上与银杏有些相似，但它缺乏银杏的许多理想治疗特性。为了防止掺假行为，实施严格的质量控制措施至关重要，包括可现场使用的快速简单的诊断工具。
本研究旨在首次开发用于快速鉴定银杏中皂荚的种特异性环介导等温扩增 (LAMP) 方法。
使用皂荚的 5.8S 部分序列和核核糖体 DNA 的内部转录间隔区 2，设计了一组四个特异性引物 (SjF3、SjB3、SjFIP 和 SjBIP) 和环引物 (SjLF 和 SjLB) 用于 LAMP 分析。
通过视觉检测检查 LAMP 分析的成功扩增，在优化条件下记录到的最高强度为 68°C 40 分钟。该引物具有高度特异性，能够准确区分皂荚和银杏以及其他 49 种药用植物。此外，所提出的 LAMP 分析证明快速、选择性和高度敏感，如绝对和相对检测限所示，分别达到皂荚 DNA 的 0.5 pg 和银杏中皂荚的 0.01%。
这种新方法允许轻松识别和区分皂荚作为银杏的潜在掺杂物，因此是质量控制的有用工具。与色谱或基于 PCR 的方法相比，该测定法证明快速、灵敏且不需要昂贵的设备，因此可能在现场分析中使用。
