Abstract
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.
Highlights
According to the World Health Organization, the use of herbal products for treating diseases is practised by 70% of the population in developing countries
The feasibility of the two DNA barcodes of ribulose biphosphate carboxylase large chain (rbcL) and internal transcribed spacer 2 (ITS2) were compared in this study
With the aid of the difference curve from the High Resolution Melting (HRM) analysis, any samples with close or similar melting temperatures could be differentiated with high resolution and power, compared to the conventional melting curve; by analysing both the melting curve shape and temperature at the same time [19,23]
Summary
According to the World Health Organization, the use of herbal products for treating diseases is practised by 70% of the population in developing countries. There are large quantities and varieties of raw materials that are sold in markets, and most of them are in processed or modified forms (dried/powdered/infusion); they are impossible to morphologically authenticate using conventional tools. This creates a challenge when trying to accurately distinguish between genuine products and counterfeit ones [1,2]. The development of an accurate, rapid, and reliable method for species identification and adulterant detection are crucial to ensure the quality and efficiency of the commercial herbal products. Realising the economic potential of the health value of herbal/medicinal
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