Herbal species authentication by melting fingerprint coupled with high resolution melting analysis (MF-HRM).

Abstract

Herbal medicines have recently attracted increasing attention for use as food supplements with health benefits; however, species authentication can be difficult due to incomplete morphological characters. Here, a molecular tool was developed for the identification of species in the National List of Essential Medicinal Plants in Thailand. The identification process used DNA fingerprints including start codon targeted (SCoT) and inter simple sequence repeat (ISSR) polymorphisms, coupled with high resolution melting (HRM), to produce melting fingerprint (MF)-HRM. Results indicated that MF-HRM, SCoT-HRM and ISSR-HRM could be used for DNA fingerprints as S34, S36, S9 and S8 of SCoT and UBC873, S25 and UBC841 of ISSR. The melting fingerprints obtained from S34 of SCoT exhibited the best primers for identification of herbal species with 87.5% accuracy and relatively high repeatability. The presence of intraspecific variation in a few species affected the shift of melting fingerprints within species. MF-HRM using S34 showed improved species prediction compared to DNA fingerprints. The concentration of DNA with 10ng/µl was recommended to perform MF-HRM. MF-HRM enabled species authentication of herbal commercialized products at only 20% resulting from the low quality of DNA isolated, while admixture of multiple product species interfered with the MF process. Findings suggested that MF-HRM showed promise as a molecular tool for the authentication of species in commercial herbal products with high specificity, moderate repeatability and rapidity without prior sequence information. This information will greatly improve quality control and traceability during the manufacturing process.