Mishra Priyanka, Shukla Ashutosh K, Sundaresan Velusamy
Plant Biology and Systematics, CSIR-Central Institute of Medicinal and Aromatic Plants, Research Centre, Bangalore, India.
Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India.
Front Plant Sci. 2018 Mar 13;9:283. doi: 10.3389/fpls.2018.00283. eCollection 2018.
(Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes ( and ) and intergenic spacers ( and ) were developed for along with the allied species. The ability and performance of the region to discriminate among the species resulted in the present proposal of the tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of species. The melting profiles of and . subsp. were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 ( crude drug sample from Bangalore) and HSA06 ( crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with . subsp. . Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate herbal products in the market for quality control in the drug supply chain. CIMAP Communication Number: CIMAP/PUB/2017/31.
豆科植物是一种全球公认的具有通便特性的药用植物，也是番泻苷的唯一来源，是印度大量出口的大宗草药。其主要采购完全来自有限的种植，这导致了有意或无意掺假的风险。市场上的原材料为粉末状或成品形式，这给鉴定带来了困难。在此，针对该植物及其近缘物种，开发了基于叶绿体基因（和）以及基因间隔区（和）的DNA条形码标签。该区域在区分该植物的物种方面的能力和表现，使得目前提出的这些标签成为成功的条形码。此外，这些标签与高分辨率熔解（HRM）曲线分析相结合，用于实时PCR基因分型方法，以衍生出Bar-HRM（条形码-HRM）分析。通过对该植物物种的基因组特征进行单核苷酸多态性（SNP）检测和突变扫描，设计了合适的HRM引物组。和亚种的熔解曲线几乎相同，其余五个物种则明显分开，因此可以通过HRM方法进行区分。该方法的灵敏度被用于鉴定市场样品[草药样品分析（HSAs）]。发现HSA01（来自班加罗尔的粗药样品）和HSA06（来自印度泰米尔纳德邦杜蒂戈林的粗药样品）被亚种高度污染。对不同百分比混合的物种掺混样品进行了灵敏鉴定，检测到的污染低至1%。PCR扩增子的熔解曲线明显不同，这使得能够通过HRM方法对物种进行可靠区分。本研究表明，DNA条形码与HRM相结合是一种有效的分子工具，可用于鉴定市场上的草药产品，以实现药品供应链中的质量控制。CIMAP通讯编号：CIMAP/PUB/2017/31。
