中药产品是中医药产业链发展的重要物质基础。中药产品制剂成型需要经过一系列加工炮制、提取制备工艺。这一系列生产工艺使得产品原料形态学性状缺失，部分化学成分变化，并引起严重的DNA降解，因此，传统的形态学、普通的化学及分子鉴定方法处理中药产品原料的鉴定问题，有时难以奏效。由于缺乏有效的鉴定手段，中药产品中原料掺假问题十分严重，阻碍了中药产业进一步发展，因此急需一种新型方法完善中药产品原料的鉴定体系。

本课题组基于Mini-barcoding技术，首次提出了中药材分子身份证这一概念。中药材分子身份证指一段极短的（20-50bp）物种特异性DNA序列。基于中药材分子身份证建立的分子鉴定方法缩短了目标序列扩增长度，极大提高了鉴定效率，可以实现目标药材的快速准确鉴定。本研究具体内容及方法如下：

基于目标药材基源物种的DNA条形码序列，建立分子身份证序列筛选数据库，通过比对分析，基于SNP位点，寻找物种特异性短序列。

改良DNA提取方法，提取中药产品的总DNA。设计特异性引物，利用PCR或恒温扩增技术（RPA）扩增含有分子身份证的短序列。

利用琼脂糖凝胶电泳、测序、试纸条等下游检测手段，对序列扩增产物进行检测，得到鉴定结果。

本研究建立了西洋参、冬虫夏草和银杏叶的分子身份证序列，实现了蒸煮物、中成药、保健品等产品中上述药材的鉴定检测。通过对西洋参、冬虫夏草蒸煮材料的实验结果可知，经处理的中药原料中的确存在严重的DNA降解，利用分子身份证技术扩增短片段，可有效缓解传统条形码技术在DNA降解严重材料中的不足。通过对人参类中成药及含有银杏叶类保健品的鉴定研究中发现，中药产品的混伪率较高，其中在24个批次的人参类中成药中，5份被西洋参替代，2份检测掺杂有西洋参；在28份银杏叶提取物中，9份检测出掺杂槐米。分别设计了冬虫夏草常见混淆品的特异引物，通过PCR方法即可检测冬虫夏草样品中是否含有混淆品。       

本研究建立了西洋参、冬虫夏草、银杏叶和槐米的分子身份证序列，结合不同扩增技术和下游检测手段，实现了中药产品原料的快速准确鉴定。经验证，分子身份证技术可发展为一种快速现场检测鉴定技术，高效鉴定中药产品中的关键及混伪原料，进而完善中药产品的监管机制。

Herbal products (HPs) are important material basis of the development of Traditional Chinese Medicine industrial chain. A series of processing technology is necessary for the preparation molding of HPs, which leads to the morphological character deficiency, chemical profile change and serious DNA degradation of raw herbal materials. Hence, traditional identification methods at morphological and chemical level or commonly-used molecular methods are sometimes difficult to be applied in the identification of raw materials in processed HPs. Due to the lack of efficient and accurate identification method; there is a very serious adulteration problem in HPs, which dramatically impede the further development of TCM industry. So it is necessary to develop an efficient molecular method for the establishment of a standard quality control system for HPs.

Based on Mini-barcoding technique, we initially proposed the conception of TCM nucleotide signature. It is an extremely short (20-50bp) and species-specific DNA sequence. By shortening amplification length of targeted sequences, the nucleotide signature methods significantly improve the identification efficiency and can rapidly identify species of TCM in HPs. The main research contents are as follows:

Firstly, database for screen of the nucleotide signature sequences was built based on DNA barcode sequences of targeted species. After sequences alignment, short and species-specific sequences were searched based on the SNP sites.

Secondly, genome DNA was extracted from HPs samples using optimized DNA extraction methods. After the design of specific primers, short sequences containing the nucleotide signature were amplified using PCR or RPA technique.

Finally, different downstream detection methods, like agarose gel electrophoresis, pyrosequencing and lateral flow stripes, were used to detect amplification results. And the identification results of HPs were obtained.

In this study, we established the nucleotide signature for Panax quinquefolius, Ophiocordyceps sinensis, and Ginkgo biloba. Using this method, we succeeded in identifying these species within decoctions, Chinese Patent Medicines and diet supplementary. According to the experiment results of P. quinquefolius and O. sinensis decoctions, it was concluded that serious DNA degradation indeed happened in processed herbal materials. And the nucleotide signature method could overcome the DNA degradation problem. According to the identification results of Chinese patent medicines containing P. quinquefolius and herbal health products containing G. biloba, the high adulteration rate was found. In the detection results, 5 samples and 2 samples of 24 different batches of P. ginseng Chinese patent medicines were respectively replaced and adulterated by unlisted P. quinquefolius; 9 samples of 28 different batches of G. biloba HPs were adulterated with unlisted S. japonica ingredients. O. sinensis and its five common adulterates can be identified using PCR methods with species-specific primers.

This study established nucleotide signature for P. quinquefolius, O. sinensis, and G. biloba. Combined with different sorts of downstream detection technique, this method can realize the rapid, accurate efficient and low-cost identification method for herbal HPs. It has been proved that the nucleotide signature method can be developed as one fast on-site identification technique to detect the key ingredients and unlisted fillers in the raw materials of HPs and further help complete the safety control system of HPs.