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.
Demand for herbal products has increased because of their purported health benefits and economic value. However, they are susceptible to adulteration, making accurate identification of herbal origin essential, especially for quality control. In recent decades, DNA-based methods have played a crucial role in the development of authentication tools that required good quality of DNA. The manufacturing process of herbal products involved heating, grinding or other mechanical procedures, and addition of excipients/additives caused DNA to degrade which in turn influenced DNA quality. In this study, nine different conventional methods with some modifications were evaluated to determine the best technique producing good quality DNA from capsule herbal products. Assessment was conducted using spectrophotometric measurements and agarose gel electrophoresis. To determine the quality of gDNA, amplification of ITS2 amplicon was performed by PCR. The DNA extraction finding showed that DNA quality from each method resulted in a different DNA purity and yield, hence the ITS2 amplification. Each of the modified DNA extraction methods performed has its own strengths and limitations when it comes to obtaining high quality gDNA. In addition, the study demonstrated the success of ITS2 amplification with the modified DNA extraction methods used.
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