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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 curve (HRM) analysis is a real-time quantitative PCR technique with outstanding advantages in rapid, sensitive and accurate detection of nucleotide differences. In recent years, gene mutation, genotyping and genetic diversity etc. The rapid development of the research, but at present the technology applied to the identification of Chinese herbal medicines less relevant reports. Based on the ITS2 sequence of plant DNA barcode, we optimized the HRM reaction system and reaction conditions by collecting 41 samples of rhizoma corydalis and its common compound adulterant. , To establish a method to detect nucleic acid fluorescence quantitative Bar-HRM of cimicifuga and its counterfeit products, and to realize identification of calamus medicinal materials and their adulterated products so as to ensure drug safety. The specificity and sensitivity of the method showed that the haplotypes of three primordial cohorts showed a good diversity, and the homozygote of both cohosh and its adulterants showed a clear distinction open. This study demonstrated that HRM has strong ability of identification of cimiental medicinal materials and their adulterants, has advantages in method universality and data visualization, and has wide application prospect.