Subject(s)
Government Regulation , Insurance Carriers , Insurance Claim Review , Humans , Insurance Carriers/legislation & jurisprudence , Insurance Carriers/standards , Insurance Claim Review/legislation & jurisprudence , Insurance Claim Review/organization & administration , Insurance Claim Review/standards , Politics , SwitzerlandABSTRACT
Admixtures of different plant species are a common problem in raw materials for medicinal use. Two exemplary assays were developed to admixtures in Helleborus niger with high-resolution melting analysis. HRM proved to be a very sensitive tool in detecting admixtures, able to detect a ratio of 1:1000 with unknown species, and of 1:200,000 with Veratrum nigrum. The example proves the ability of HRM for quantification in multiplex PCR. The method is not limited to detecting adulterations. It can also be used to quantify a specific target by integrating a second amplicon in the assay as internal standard.
Subject(s)
DNA, Plant/chemistry , Polymerase Chain Reaction/methods , DNA Primers/chemistry , Genotype , Helleborus/genetics , Nucleic Acid Denaturation , Spectrometry, Fluorescence , Transition Temperature , Veratrum/geneticsABSTRACT
Hellbori nigri rhizoma is a drug that is difficult to distinguish from other species of the genus Helleborus. In this communication we present a DNA-based identification by high-resolution melting analysis (HRM) that is able to differentiate between Helleborus niger and other species of the genus. HRM is a very specific, time- and labour-saving method for identifying DNA sequence variations and is ideally suitable for routine PCR analysis. The HRM assay developed is specific for the genus Helleborus. This method not only detects the presence of the target species H. niger but also, to a certain extent, identifies other Helleborus species by their different melting curve shapes. Markers were developed based on the trnL-trnF intergenic spacer and on the matK sequence. For an unambiguous identification of Helleborus niger, melting curves of both markers should be used.
Subject(s)
DNA, Plant , Drug Contamination/prevention & control , Genetic Variation , Helleborus/genetics , Polymerase Chain Reaction/methods , Base Sequence , DNA, Intergenic , Freezing , Genes, Plant , Genetic Markers , Plants, Medicinal , Species SpecificityABSTRACT
BACKGROUND: High resolution melting curve analysis (HRM) is a technique that measures exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double stranded DNA. The measurement is immediately following PCR in a one-step, closed-tube method. The shape of the melting curve depends on the GC content, length and sequence of the amplicon. Hence it is a powerful, fast and cheap method to detect Single Nucleotide Polymorphisms (SNPs) and other mutations. RESULTS: Here we present a strategy to set up microsatellite analysis for HRM including the correct assignment of heterozygous samples by comparative analysis and artificial mixtures of samples. The approach is demonstrated on two Simple Sequence Repeat (SSR) loci of different complexity in the genus Origanum. Following this strategy all alleles of our sample sets could be classified correctly. CONCLUSION: HRM can be used in microsatellite analysis and other codominant marker systems implementing a protocol of comparative melting curve assignment with artificial mixtures of samples to overcome difficulties in correctly assigning heterozygous samples. The method is faster, more sensitive and cheaper than standard protocols for microsatellite analysis.
