Centella asiatica (L.) Urb., used in pharmaceuticals, cosmetics, and herbal medicines, is mainly cultivated in open fields. The fact that it shares morphological traits and ecological environment with Hydrocotyle species and another herbal medicine, Glechoma longituba (Nakai) Kuprian raises concerns about its misuse and contamination. Diagnostic assays capable of effectively discriminating impurities are crucial for consistent quality control of industrial raw materials. Therefore, we aimed to establish a rapid, accurate, and highly sensitive genetic discrimination method using dominant sequence-characterized amplified region (SCAR) markers and conventional polymerase chain reaction (PCR). Six species, namely C. asiatica , four Hydrocotyle species ( H. maritima Honda, H. ramiflora Maxim., H. yabei Makino, and H. javanica Thunb.), and G. longituba , were studied. Comparative sequence analysis and phylogeny based on internal transcribed spacer (ITS) barcoding confirmed that the region was a useful genetic marker for identification for these plant species. The SCAR primers were designed by positioning species-specific nucleotides at the 3′ end and incorporating deliberate nucleotide substitutions to enhance discrimination as dominant markers. In this study, we designed SCAR primer sets targeting amplicons <450 bp for the SCAR PCR assay. The limit of detection of up to 10 pg (0.1%) of individual DNA was demonstrated for all SCAR markers. This demonstrated that the assay is effective for genetic authentication of C. asiatica in dried materials with degraded DNA, including large-scale industrial raw materials.
Kim et al. (Fri,) studied this question.