基于DNA metabarcoding技术的中成药金嗓清音丸基原物种鉴定研究

Species identification in Chinese patent medicine is a difficulty in the study of traditional Chinese medicine identification. How to identify species is becoming one of the major factors to hinder the establishment identification system and quality control in Chinese patent medicine. We selecte Jinsang Qingyin Wan (JSQYW), a classical herbal prescription, containing Dendrobii Caulis, Fritillariae Cirrhosae Bulbus and Bombyx Batryticatus et al, as the model for the application of the herbal species composition monitoring method through metabarcoding and high-throughput sequencing. Reference experimental mixtures and commercial JSQYW products from the marketplace were used to confirm the method. Firstly, samples were treated by DNA extraction and Illumina sequencing platform to construct a PCR-free library. Secondly, samples were treated by DNA extraction, DNA amplification and PCR product purification to construct an SMRT sequencing library. To distinguish between PCR amplicons derived from different samples, another short sequence tag were designed and added to the 5′end of the universal primers of the ITS2, psbA-trnH and COI sequences. Finally, the results suggest that with the combination of metabarcoding and high-throughput sequencing, it is reliable and sensitive enough to detect species in the JSQYW, and adulterants/contaminants. Taken together, the study will provide theoretical and experimental basis for becoming a valuable tool for the biomonitoring of multi-ingredient Chinese patent medicine.

中成药基原物种鉴定是中药鉴定研究的难点。如何溯源中成药物种成分组成，已成为阻碍建立中成药鉴定体系和实现中成药品质控制的技术瓶颈。本项目拟以含有石斛、川贝母、僵蚕等处方药材混伪掺假突出的中成药金嗓清音丸为切入点，首先对处方单味药材进行DNA条形码分子鉴定，保证原药材的准确，并自制对照样品，利用二代、三代高通量测序平台联合DNA metabarcoding构建分子鉴定体系，其次将该体系应用于市售不同批次金嗓清音丸。分别完成样品DNA提取，基于Illumina平台构建PCR-free文库；在通用引物ITS2、psbA-trnH和COI的5’端设计短序列标签，优化PCR扩增和产物回收方法，基于PacBio SMRT测序平台建库；最后分别进行测序数据生物信息学分析，溯源金嗓清音丸物种组成，阐明其是否存在混伪掺假问题。完成本项目为中成药准确鉴定和质量控制提供新思路，为临床安全用药提供科学依据和参考。

中成药基原物种鉴定是中药鉴定研究的难点。如何溯源中成药物种成分组成，已成为阻碍建立中成药鉴定体系和实现中成药品质控制的技术瓶颈。本课题中，我们以含有石斛、川贝母、僵蚕等处方药材混伪掺假突出的中成药金嗓清音丸为切入点，将DNA条形码鉴定、宏基因组测序和单分子测序等关键技术应用于中成药鉴定领域。首先对金嗓清音丸处方单味药材进行DNA条形码分子鉴定，保证原药材的准确，并自制对照样品，利用二代、三代高通量测序平台联合DNA metabarcoding构建分子鉴定体系，其次将该体系应用于市售不同批次金嗓清音丸。首先，基于二代Illumina Miseq测序平台的DNA metabarcoding进行宏基因组测序，建立PCR-free文库，进行生物信息学数据分析，获得高质量测序reads比对ITS2、psbA-trnH、matK、rbcL和COI数据库，在中药材DNA条形码鉴定系统进行物种鉴定分析。其次，联合二代Illumina Miseq和三代PacBio SMRT测序平台的DNA metabarcoding金嗓清音丸鉴定体系，通过磁珠法提取中成药DNA，按指定测序区域，合成带有barcode的特异引物，PCR扩增，PCR扩增产物胶回收纯化，DNA质量检测，文库构建及上机测序。Miseq测序得到的PE reads首先根据overlap关系进行拼接，同时对序列质量进行质控和过滤；PacBio测序得到的reads首先根据CCS算法得到一致性序列，同时对序列质量进行质控和过滤，区分样本后进行OTU聚类分析和物种分类学分析。将所得ITS、ITS2、psbA-trnH和COI序列在中药材DNA条形码鉴定系统中进行物种鉴定。研究表明联合新一代测序技术和DNA条形码技术的方法可成功应用于中成药金嗓清音丸的物种组成分析，为中成药金嗓清音丸的质量评价提供科学基础。对推进中药鉴定走向标准化、数字化和网络化，建立从源头到产品的标准化鉴定流程，并推进其纳入《中国药典》中成药质量监测标准，与国际接轨，推动中医药走向世界，具有重要意义。