高分辨率熔解曲线技术在多基原药材升麻鉴定中的应用

Application of high resolution melting curve to identification of Cimicifugae Rhizoma

高分辨率熔解曲线(HRM)分析是一种实时定量PCR技术,在核苷酸差异检测中具有快速、灵敏和准确等突出的优点,近几年来在基因突变、基因分型和遗传多样性等研究中发展迅速,但目前该技术应用于中药材鉴定的相关报道较少。本研究选取多基原药材升麻作为研究对象,广泛收集其主产区药材基原及其常见混伪品共41个样品,基于植物DNA条形码ITS2序列,通过对HRM反应体系和反应条件的优化,建立检测升麻及其混伪品的核酸荧光定量Bar-HRM方法,实现对升麻药材及其混伪品的鉴定,从而确保用药安全。该方法的特异性和灵敏性研究结果显示,升麻3个基原的各单倍型均能很好地表现出差异性,升麻及其混伪品均表现出良好单系性,能明显区分开。本研究证明HRM技术对升麻药材及其混伪品的鉴定能力强,在方法通用性和数据可视化方面具有优势,应用前景广阔。

High-resolution-melting analysis （HRM） is a new technology derived from qPCR and is widely used in the study of polymorphism, genotyping, and single nucleotide mutation. Advantages of HRM include cost-effectiveness and time-efficiency over PCR-based genotyping. However, the application of HRM in the authentication of herbal products is still limited with few studies on the classification and identification of herbal products. In this study, Cimicifugae Rhizoma was used as an example to verify the stability and accuracy of HRM technique in identification of Chinese materia medica. HRM assay was established for identification based on ITS2 region of Cimicifugae Rhizomas and its adulterants （including 41 samples）. Our findings showed that HRM allows not only the identification of adulteration but also the quantification of the most common admixture. This study is significant for better quality in the verification of the authenticity of herbal medicine. The method is promising for future identification of traditional Chinese medicinal materials.