基于二代高通量DNA测序技术的混合中草药物种鉴定研究

Mixed herbal products are very popular in the market, however, the good and the bad are intermingled. Due to make with multi-herbal powder, it is hardly to accurately identify the herbal species in mixed herbal products by traditional morphology, physical or chemical methods, and even conventional DNA barcoding exist great limitations to identify multi-herbal samples. Therefore, it is urgently need to develop an accurate and efficient approach to identify the species of herbal ingredient in mixed herbal products. In this study, we choose commercial multi-herbal tea products as the object, extract genomic DNA, analysis the ITS2 and psbA-trnH sequences using the Illumina MiSeq high-throughput DNA sequencing, and then align the obtained sequences to the reference ones in the traditional Chinese medicine database and finally achieve the species identification of multi-herbal tea products. This method can not only identify herbs listed on the product labels, but also detect medicine substitution and contamination in products. Thus, this study will provide a high-efficiency, low-cost herbal ingredient identification approach for mixed herbal products, and ensure the quality and safety.

混合中草药产品在市面上极为普遍，然而品质良莠不齐。由于这些产品由多种中药材粉末混合制成，用传统形态、理化鉴定难以对其原料药材的物种进行准确鉴定，而常规的DNA条形码技术在解决多种混合药材鉴定时也存在很大的局限性，因此亟需开发准确、高效的混合中草药物种鉴定方法。本研究拟以市售混合草药茶为研究对象，提取DNA，应用IlluminaMiSeq二代高通量DNA技术对ITS2和psbA-trnH序列进行测序，通过与构建的中草药DNA条形码数据库中序列比对分析，实现对混合中药材制品中每一味中草药原料的物种鉴定。该方法不仅能准确鉴定混合中草药产品中标识的中草药物种，同时能准确检测出产品的药材混伪、掺杂等质量问题，将为混合药材制品的原料物种鉴定提供一种高效、便捷、低成本且便于应用的新方法，保障其质量和食用安全。

市售混合中药材产品良多，然品质良莠不齐，其原料中药材物种鉴定困难，尚缺乏准确有效的鉴定方法。本项目以混合草药茶为例，收集常用的草药茶药材87种，新增常见草药茶原料物种及其混伪品ITS2序列558条，建立常用草药茶药材的DNA条形码数据库，为草药茶原料物种的准确鉴定提供了准确的数据基础。在此基础上，完成Illumina MiSeq二代高通量DNA测序技术用于混合药材原料物种鉴定的方法摸索和技术评价，结果表明MiSeq二代高通量测序技术在混合药材鉴定中通量高、检出率高、检出深度可达1%，且成本低，能够高效检出混合药材中的原料物种。同时，初步建立了Miseq高通量技术用于混合药材鉴定原料及掺杂掺假物种鉴定的技术流程，为混合中药材产品的原料溯源和质量控制提供新的鉴定方法。