基于HRM的常见伞形科香辛料快速鉴定

Rapid Identification of Common Umbelliferae Spices Based on HRM

目的：4种香辛料小茴香、孜然、葛缕子、莳萝子与其混伪品蛇床子、防风子因外形特征相似,易混淆使用,带来安全隐患。针对鉴别困难的问题,建立高分辨率熔解曲线（HRM）分析法,以实现简便、快速鉴别的目的。方法：收集这4种香辛料及常见混伪品蛇床子、防风子共23份样本,作为参照样本,提取总DNA,基于内转录间隔区2（ITS2）序列通过聚合酶链式反应（PCR）进一步确定物种,并应用HRM技术建立检测方法和熔解曲线模型。香辛料市场收集这4种香辛料共17份样本,作为市场样本,通过HRM技术鉴定物种是否与标签相符。结果：小茴香、孜然、葛缕子、莳萝子、蛇床子、防风子的熔解曲线均表现出良好差异性,能明显区分开;17份市场样本中,编号N1和N3样品熔解曲线与标签显示的莳萝子熔解曲线具有明显差异,进一步测序结果显示二者均为毒芹子。结论：HRM技术能实现小茴香等伞形科香辛料及其混伪品间的可视化鉴别,可作为DNA条形码技术的有力补充,在中药资源鉴定中应用前景广阔。

Objective： Desiccative ripe fruits of Foeniculum vulgare,Cuminum cyminum,Anethum graveolens,Carum carvi,Cnidium monnieri and Saposhnikovia divaricata were used as the research objects,these fruits had similar appearance and they are easy to be confused,which will give rise to security risks. To develop a high resolution melting curve（ HRM） analysis method in a fast and easy way targeted on the problem of identification of these medicinal materials. Method： Detection method and melting curve model was established for identification based on internal transcribed spacer 2（ ITS2） region of the four spices and their common adulterants of C. monnieri fruit and S. divaricata fruit,there were 23 reference samples. Then the four spices（ including 17 market samples） were collected form spice market,and they were identified if the species was compatible with the label by HRM. Result： The melting curves of the four spices and their adulterants had clear diversity. The two of the 17 market samples,N1 and N3,were potentially mislabeled,and their sequencing results showed that both were Cicuta virosa. Conclusion： HRM technology can realize the visual identification of common Umbelliferae spices and their adulterants,and it can be used as a powerful supplement of DNA barcoding technology.