以人参属中成药为例探讨基于Meta-barcode和Mini-barcode相结合鉴定中成药的可能

The DNA barcoding system of Chinese herbal medicine developed by our research group is widely considered as a technology revolution that realize the transition from the empirical identification process mostly based on the appearance and taste of herbs to an objective process based on universal molecular markers, which provide powerful tools to realize the identification of seeds, seedlings, raw plants and Chinese traditional medicines (including medicine slices) and solve industrial problems such as herb mixture or adulteration. However, this system has not involved identifications of Chinese patent medicine so far and cannot realize all aspects of monitoring in the productive processes of Chinese patent medicine. In this research, ten sorts of Chinese patent medicine containing Panax genus are the main study objects including all main dosage forms such as pills, capsules, slices and powders. Meta-barcoding of Chinese patent medicine is conducted using universal primer pair 2F/3R. Aimed at materials with severe DNA degradation, extractives for example, the applicant initially proposed the nucleotide signature method that are based on the analysis of large sample size of barcode sequences and SNP sites to select one species- specific nucleotide signature of length 20bp to 50bp for each single herb. The screening process involves python programming, searching within Entrez database, alinement by MUSCLE method, finding conserved sites with CodonCode software, design of specific primer pares for nucleotide signatures and amplification of mini-barcoding sequences containing nucleotide signatures. We aim to establish a Chinese patent medicine identification platform based on the second generation sequencing barcoding technology combined with Meta-barcode and Mini-barcode to systematically analyze the ingredients of Chinese patent medicine. This research will definitely provide a significant basis for the tracing of DNA barcoding for seeds, seedlings, raw plants and Chinese traditional medicines and completely eradicate the fake medicine problems.

课题组已建立的中草药DNA条形码体系为解决中草药混用和掺假等行业问题提供了强有力工具，可以实现对种子种苗-原植物-中药材（饮片）的鉴定，但目前尚未涉及中成药的鉴定，无法实现中药生产过程中的全方位监测。本研究以十种人参属中成药为研究对象，基于二代测序对中成药进行Meta-barcode鉴定；对于提取物等DNA降解严重的材料，申请者创新性提出了Mini-barcode方法，筛选20-50bp的分子身份证用于鉴定，通过Python编程；借助Entrez数据获取系统；Muscle进行序列比对；CodonCode查找保守位点及SNP（s）；设计特异引物扩增带有分子身份证的Mini-barcode序列。搭建Meta-barcode和Mini-barcode相结合的中成药鉴定平台，全面解析中成药组分。本研究将对实现从种子种苗-原植物-中药材（饮片）-中成药的DNA条形溯源提供重要依据，使得假药无处可藏。

本研究建立了Meta-barcode和Mini-barcode鉴定中成药的方法，为解决中成药掺伪或误用问题提供了技术保障。首次提出应用20-40bp的分子身份证鉴定中成药，为使研究方法可靠，研究材料不仅涉及了包括人参属中成药还包括市售常见的多种中成药，如当归、冬虫夏草、肉苁蓉、金银花、杜仲、银杏、川贝母中成药等。首次提出双峰法鉴定混合粉末，成功应用于人参和西洋参、金银花和山银花的中成药鉴定。相关研究成果已发表在国内外杂志上。本研究将对实现从种子种苗-原植物-中药材（饮片）-中成药的DNA条形溯源提供重要依据，使得假药无处可藏。