Department of Chinese Pharmaceutical Science and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan.
Department of Medicinal Botanicals and Foods on Health Applications, Da-Yeh University, Changhua 515006, Taiwan.
Int J Mol Sci. 2023 Jun 27;24(13):10730. doi: 10.3390/ijms241310730.
(PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named "loop-mediated isothermal amplification (LAMP)" was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products.
（PO）是一种常见的药用作物，因其作为蔬菜在饮食中的应用，是中药（TCM）的重要成分。PO 已被记录在草药市场中经常被其他相关物种掺假，从而扭曲了 PO 植物的身份。因此，在进行药物或功能性食品应用之前，PO 的植物来源鉴定是至关重要的一步。在这项研究中，建立了一种名为“环介导等温扩增（LAMP）”的快速检测方法，用于 PO DNA 的特异性和敏感鉴定。基于 PO 和掺杂物种在内部转录间隔区 2（ITS2）序列上的差异，设计了 LAMP 引物，并验证了它们的特异性、敏感性和用于 PO DNA 鉴定的应用。LAMP 检测 PO DNA 鉴定的检测限在 63°C 等温条件下为 30 分钟时，特异性为 100 fg。此外，不同热处理的 PO 样品可应用于 LAMP 检测进行 PO 鉴定。与煮沸和干燥处理相比，这些 PO 样品更易受到蒸汽处理对鉴定的影响。此外，从草药市场采购的商业 PO 样品用于展示开发的 LAMP 分析对 PO 收获后鉴定的适用性，调查发现，草药市场上约 68.4%的 PO 标本存在掺假情况。总之，本文首次成功开发了用于 PO 鉴定的特异性、敏感性和快速 LAMP 检测方法，并展示了其在草药市场 PO 样品掺假检测中的实际应用。本研究中创建的 LAMP 检测方法将有助于 PO 及其商业产品的植物来源鉴定。
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