Overcoming Challenges in DNA Extractions from Triphala Ingredients: A Way Forward for Optimization of Conventional and Digital PCR Assays for Molecular Authentication

Abstract

Terminalia bellirica (TB), Terminalia chebula (TC), and Phyllanthus emblica (PE) fruits are renowned for their diverse therapeutic benefits, propelling their cultivation and use in herbal remedies. However, the global surge in demand driven by the awareness and long-term benefits of using herbal medicines has inadvertently led to a rise in adulteration practices within the herbal market. Recent advancement in DNA authentication of herbal products is constrained by poor quality and quantity of PCR amplifiable DNA obtained from the dried and polyphenol-rich fruits of processed herbal products, resulting in inconsistent PCR amplification due to heterogeneous secondary metabolites. This study tailored a DNA isolation protocol by optimizing buffering strength to stabilize pH and adding phenolic compound scavenger additives, such as polyvinylpyrrolidone, during the cell lysis step. The implemented procedure resulted in significant enhancements in both the quantity and quality of PCR amplifiable DNA. PCR amenability was evaluated using ITS2 metabarcode. Later, species-specific assays, targeting ITS-based SCAR markers specific to TB, TC, and PE, were performed on six market powders for each plant species. TB, TC, and PE were detected in 100, 83.3, and 50% of the six market samples, respectively. Digital PCR increases the assay’s sensitivity by two-fold compared to conventional PCR. To the best of our knowledge, this is the first instance of utilizing dPCR for authenticating TB, TC, and PE fruits. The improvised DNA extraction protocol successfully demonstrates how a comprehensive analysis of PCR amplifiable DNA isolation and PCR dynamics enables the effective resolution of challenges related to poor DNA quality and quantity, as well as the inconsistency encountered during PCR due to the heterogeneity of polyphenols.