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Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis

机译:棒-高分辨熔解法检测商品草药中长生杜仲的真伪

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摘要

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.
机译:本研究表明高分辨率熔解(HRM)分析与DNA条码(Bar-HRM)结合使用,可作为一种快速,灵敏的技术来检测长叶杜仲商业草药产品中的掺假物。针对叶绿体区域-核糖二磷酸羧化酶大链(rbcL)和核糖体区域-内部转录间隔区2(ITS2)的DNA条形码,使用饱和的Eva绿色染料作为荧光信号源完成了PCR扩增和HRM分析。通过使用实时循环仪。通过测序,从Genbank数据库中鉴定未知序列,并使用邻居联合(NJ)分析生成系统树,进一步验证了结果。两种DNA标记均显示出可区分的融解温度和参比与掺假物之间标准化曲线的形状。在物种识别的情况下,ITS2在区分物种方面更为成功。此外,检测到的混合样品中含有少量痕量的靶标长叶大肠杆菌DNA(w / v),对于rbcL的检测可低至5%,而对于ITS2的检测则小于1%,证明了HRM分析的灵敏度和多功能性。总之,Bar-HRM分析是一种快速而可靠的技术,可以有效地检测草药产品中的掺杂物。因此,这对于监管机构管理食品安全问题将是有益的。

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