基于LAMP和特异性PCR扩增技术沉香即时鉴定分子标志物谱的研究

Agarwood is rare medicinal materials and perfume, one of the most important spices. It is used in two thousand prescription of traditional Chinese medicine or Tibetan medicine in China. There are 26 species of plants in Aquilaria Lam. and Gyrinops spp., an endangered in the family Thymelaeaceae, which can be used for the production of agarwood. But "Pharmacopoeia of the people republic of China" provisions of domestic medicinal materials as Aquila sinensis (Lour.) Gilg fragrant resinous wood. Demand for agarwood is high in the international market due to its scarcity, which cause the adulteration is very serious. In the agarwood industry, consumers' preference is often influenced by the geographical origin of the agarwood,which is generally indicative of the species growing in that region and hence the supposed ‘quality’ of an agarwood product. Therefore, this study will screening strong specificity and high singleness of 26 types molecular markers in agarwood, using specific fragments of plastid genes and nuclear genes of genera , based on LAMP and specific PCR amplification technique. It is combined with fluorescence color to achieve instant visual identification of agarwood. The results of the study as a technical means for rapid identification of the authenticity and international trade customs of agarwood.

沉香为名贵药材在我国2000个中药或藏药组方中使用，作为香料居“沉檀龙麝”四大名香之首。瑞香科沉香属及拟沉香属26种植物均可产沉香，但《中国药典》仅收载白木香Aquilaira sinensis (Lour.) Gilg.所产沉香。沉香因市场需求大，掺杂使假现象极为严重。因此，本研究将基于LAMP和特异性PCR扩增技术，研究以属、种为分类层级的质基因和核基因的特异性片段，筛选涵括26种沉香植物且特异性强、专一性高的种属分子标志物谱，并结合荧光显色实现对沉香即时可视化鉴定，作为沉香药材真伪快速鉴定和国际贸易海关执法的技术手段。

沉香为名贵中药和香料，目前沉香属植物约有19个种，但流通于市场上的法定临床药用沉香只有国产白木香Aquilaria sinensis和进口沉香Aquilaria malaccensis 2种。贸易市场上沉香药材混乱，不法商贩为了牟取暴利，在沉香中掺杂使假或替代的现象屡见不鲜，出现临床用药安全隐患。此外，因沉香DNA降解严重，难以像植物DNA那样，从样品DNA提取、扩增、测序及分析过程，耗时较长，在实际使用条形码组合效果不佳。因此，本项目主要开展筛选特异性PCR扩增技术及标志物，建立快速、高效的鉴别方法。通过实施，本项目已经建立了沉香药材的高质量DNA提取方法，并利用高度树脂化沉香、沉香加工残留物、不同沉香物种的材料商进行了验证；以属、种为分类层级的质基因和核基因的特异性片段标志物的筛选，通过使用LAMP扩增技术筛选等到了沉香与非沉香的快速鉴定标志物，能够快速从样品中筛选出沉香属的物种，通过使用Bar-HRM技术筛选得到了沉香属内主要物种鉴别的特异性标志物，能够从样品中鉴定出药用沉香及主要产区的沉香物种。同时，针对现有数据库中沉香属的序列，分析得到了产沉香物种的ITS2特异性二级结构。研究结果有助于不同物种沉香及伪品的简便、准确、快速识别，使其成为分子鉴定最有利的工具，为沉香国际贸易中CITES管理和海关执法等提供重要科学依据，进而明确药用沉香物种信息，解决临床上用药安全问题。