以金龙胶囊为例探讨不依赖PCR的宏基因组技术一步鉴定中成药的可能

Currently, counterfeit Chinese patent medicine products containing fake crude drugs are frequently found. It’s very difficult to make a clear inspection. Although encouraging progress has been made in recent years in the molecular identification of crude drugs, its application in Chinese patent medicine remains a challenging work. So far in the studies concerning analysis of biological ingredients in Chinese patent medicine products, PCR amplification of certain barcode regions followed by Sanger sequencing or high throughput sequencing (HTS) comprises a usual procedure; but that’s often blamed for low identification efficiency and massive unrelated species information. There are two probable reasons: the one is that the difference of primer universality and amplification efficiency across species may lead to the lost of some species’ data, the other is that PCR biases may easily be detected by HTS and leads to biodiversity inflation. Recently, PCR-independent metagemomics showed promise as a solution to above mentioned problems; its application in biodiversity investigation of a bulk insect sample pooling 49 species proved to be perfect with both efficiency and accuracy of 100%. The applicants are to make an exploration of this technique in Chinese patent medicine identification. Jinlong Capsule, an anticancer patent medicine comprised of fresh body of Gekko spp., Bungarus multicinctus, and Deinagkistrodon acutus, and prepared through freeze drying, is chosen as an model to be studied. After DNA extraction and normalizing, ultra-deep sequencing through an Illumina Hiseq 2000 platform is to be performed; de novo assembly and annotation with proper bioinformatics software will give whole mitochondrial genome of each ingredient, and species assignment and identification will be done based on the mitogenomes. The research will provide a new template for molecular identification of Chinese patent medicine.

目前中成药造假多发，虚假投料常见，鉴别困难。中药分子鉴定近年取得了令人鼓舞的进展，但中成药的分子鉴定仍是一项具有挑战性的工作。现有对中成药组分全面鉴定的研究，均采用PCR扩增某些基因片段，进行克隆测序或高通量测序；但通常鉴定效率低，并有大量未经验证的无关物种信息。究其原因，一是混合样品中物种间引物通用性和扩增效率不同，易发生物种丢失；二是扩增误差易被高通量测序检出，造成多样性膨胀。最近，用不依赖PCR扩增的宏基因组技术，对49种昆虫混合而成的样品中物种鉴定的研究，成功避免了上述问题，效率和准确率为100%。申请者拟探索此技术用于中成药鉴定的可能。以由鲜守宫、鲜金钱白花蛇、鲜蕲蛇经冷冻干燥等工艺制备的抗癌中成药金龙胶囊为研究对象，提取DNA后用Illumina平台进行超深度测序，用合适的软件进行从头组装及注释，获得各组分的线粒体基因组序列，鉴定其物种来源。项目可为中成药分子鉴定提供新方法。

目前中成药造假多发，虚假投料常见，鉴别困难。中成药的分子鉴定仍是一项具有挑战性的工作。现有对中成药组分全面鉴定的研究，均采用PCR扩增某些基因片段，进行克隆测序或高通量测序；但通常鉴定效率低，并有大量未经验证的无关物种信息。究其原因，一是混合样品中物种间引物通用性和扩增效率不同，易发生物种丢失；二是扩增误差易被高通量测序检出，造成多样性膨胀。近年，用不依赖PCR扩增的宏基因组技术，对49种昆虫混合而成的样品中物种鉴定的研究，成功避免了上述问题，效率和准确率为100%。项目组拟以由鲜守宫、鲜金钱白花蛇、鲜蕲蛇经冷冻干燥等工艺制备的抗癌中成药金龙胶囊为研究对象，探索此技术用于中成药鉴定的可能。.项目实施期间，项目组采集和收集了金龙胶囊三种生物组分的正品和混伪品原动物30种40余份，建立了含壁虎下目、蝰科、眼镜蛇科、游蛇科100个物种200条线粒体全基因组序列的数据库。将无蹼壁虎、尖吻蝮、银环蛇的DNA按2:1:1混合后进行高通量测序，从中完整组装出三者的线粒体基因组。进一步地，按照质量比2:1:1，使用原动物的肌肉组织，自制原材料掺伪金龙胶囊样品YP-1（无蹼壁虎200mg：尖吻蝮100mg：赤链蛇100mg）和YP-2（无蹼壁虎200mg:银环蛇100mg:金环蛇50mg+赤链蛇50mg），提取DNA后进行高通量测序，亦成功组装、注释出各动物的完整线粒体基因组。接下来探索了从市售金龙胶囊中提取DNA的方法，以改良SDS法为好；然而高通量测序尚未能取得满意结果，有待完善。.另外，项目组探讨了用分子定量法对金龙胶囊进行质量控制。发现物种特异性片段的拷贝数能够反映原料的投料量；测定了市售金龙胶囊中蕲蛇片段的拷贝数，提出了建议性的质控标准；首次对鲜、干金钱白花蛇进行了分子定量鉴别，设定了鉴别标准。.项目可为中成药中的生物组分鉴定和质量控制研究，特别是含动物类生药的中成药，提供参考。