利用多重连接探针扩增结合熔解曲线法鉴别五味子与南五味子掺杂

Identification of Inter-Adulteration of Schisandra Chinensis and Schisandra Sphenanthera by Multiplexed Ligation Dependent Probe Amplification Combined with Melting Crave Method

目的探究多重连接探针扩增技术(MLPA)结合熔解曲线在五味子和南五味子鉴别中的应用。方法以五味子和南五味子为研究对象,基于其ITS2序列设计探针进行MLPA反应,通过分析荧光熔解曲线的Tm值对二者进行鉴别,并将此方法应用到市场随机收集的31份五味子和南五味子商品中,通过对聚合酶链反应(PCR)产物测序验证此方法的准确性。结果两对探针都表现出很好的特异性和灵敏度,均只出现对应的单一熔解曲线峰,五味子产生82.3℃的熔解曲线峰,南五味子产生80.7℃的熔解曲线峰。31份五味子商品中,16份商品的熔解曲线Tm值与五味子吻合,15份商品的熔解曲线Tm值与南五味子吻合,实验结果与测序结果一致。结论该研究建立的方法特异性强,灵敏度高,且操作简单方便,可以快速鉴别五味子及其掺混品。

Objective To explore the application of multiplex ligation-dependent probe amplification(MLPA)combined with a melting curve in identifying Schisandra chinensis and Schisandra sphenanthera.Methods Based on the ITS2 sequence of Schisandra chinensis and Schisandra sphenanthera,the probe was designed for MLPA reaction.The Tm value of the fluorescence fusion curve was analyzed to identify the two species.The method was applied to 31 Schisandra chinensis and Schisandra sphenanthera products randomly collected from the market,and the method's accuracy was verified by PCR product sequencing.Results The two pairs of probes showed good specificity and sensitivity,and only the corresponding single melting curve peak occurred in each pair.Schisandra chinensis produced a melting curve peak of 82.3℃,and Schisandra sphenanthera produced a melting curve peak of 80.7℃.Among the 31 Schisandra products,the Tm value of the melting curve of 16 products was consistent with that of Schisandra chinensis,and the other 15 products were consistent with that of Schisandra sphenanthera.The experimental results were consistent with the sequencing results.Conclusion The method established in this study has strong specificity,high sensitivity,simple and convenient operation,and can quickly identify Schisandra chinensis and its mixtures.