Rapid Authentication of Flowers of Panax ginseng and Panax notoginseng Using High-Resolution Melting (HRM) Analysis

The flowers of Panax ginseng C. A. Mey. (PG) and Panax notoginseng (Burkill) F. H. Chen ex C. H. Chow (PN) are morphologically indistinguishable after drying, leading to prevalent adulteration that compromises product quality and consumer safety. To address this issue, we developed a rapid, closed-tube molecular authentication method based on high-resolution melting (HRM) analysis. Species-specific primer pairs were designed to target the conserved ITS and rbcL-accD regions, with PNG-2 selected as the optimal candidate owing to its stable genotyping performance and moderate GC content. Our results established GC content, rather than amplicon length, as the primary determinant of the melting temperature (Tm). Notably, the experimentally measured Tm values were consistently 0.7-1.5 °C higher than theoretical predictions, a discrepancy attributable to the stabilizing effect of the saturated fluorescent dye. To ensure maximum diagnostic reliability, the HRM results were cross-validated through a three-tier system comprising ITS2 phylogenetic analysis, agarose gel electrophoresis, and Sanger sequencing. The practical utility and matrix robustness of the assay were further verified using a diversified validation cohort of 30 commercial samples, including 24 floral batches and 6 root-derived products (root slices and ultramicro powders). The HRM profiles demonstrated 100% concordance with DNA barcoding results, effectively identifying mislabeled products across different botanical matrices and processing forms. This methodology, which can be completed within 3 h, provides a significantly more cost-effective and rapid alternative to traditional sequencing-based methods for large-scale market surveillance and industrial quality control.