基于PCR技术及G-qudruplex比色探针的藏成药中藏红花可视化基因检测体系的建立及应用

藏红花（Crocus sativus L.）是一种在复方成药制剂中应用广泛的贵重民族药材，指标成分为藏红花素类（Crocin），但是该类成分还存在于栀子等药材，采用HPLC等化学分析方法难以确认复方成药制剂中藏红花投料的真实性。在前期的研究中，申请人发现采用可视化基因检测方法可以实现对包括片剂、丸剂、口服液、注射液等剂型在内的复方成药制剂的真伪进行鉴别。在此基础上，针对复方藏成药制剂中藏红花检测困难的情况，在藏红花的鉴定中引入可视化基因检测方法，以含有藏红花的多种藏成药（如八味西红花止血散、红花如意丸）为研究对象，利用聚合酶链式反应（PCR）对差异性大、拷贝数高的ITS序列进行特异性扩增，并将PCR扩增与新型的G-qudruplex（G-四聚体）比色探针相结合，构建出一个准确、快捷、成本合理的藏成药中藏红花的可视化基因检测体系，为成分复杂、药材经加工的藏成药中贵重药材的鉴定探索一条新的道路。

Crocus sativus is one of the widely used valuable regional medicinal materials in compound preparations. It can really be misleaded only to detect the Crocin in processed medicine by HPLC as Crocin is also found in Gardenia jasminoides Ellisthis and other medicinal plants. Our previous study shows that we can apply a DNA based colorimetric detection method to the identification of CPM in the form of tablets, pills, oral liquids and also injections. On the basis of this study, a colorimetric gene test is introduced for the difficulties of the authentication of Crocus sativus in complex preparations. Selecting several kinds of Crocus sativus contained TPM（Tibetan patent medicine)（Ba wei xi hong hua zhi xue san and Hong hua ru yi wan）as the research object, the species specific and highly repetitive ITS sequence will be amplified by PCR (Polymerase Chain reaction). Furthermore, the combination of PCR and G-qudruplex colorimetric probes will finally make it an accurate、fast and cost-effective detection system of Crocus sativus in TPM and it can also provides a new way to detect other precious medicinal materials in complex and processed Tibetan patent medicine.