High-Resolution Melting Analysis as a Tool for Plant Species Authentication.

Abstract

High-resolution melting (HRM) analysis is a cost-effective, specific, and rapid tool that allows distinguishing genetically related plants and other organisms based on the detection of small nucleotide variations, which are recognized from melting properties of the double-stranded DNA. It has been widely applied in several areas of research and diagnostics, including botanical authentication of several food commodities and herbal products. Generally, it consists of the main steps: (1) in silico sequence analysis and primer design; (2) DNA extraction from plant material; (3) amplification by real-time PCR with an enhanced fluorescent dye targeting a specific DNA barcode or other regions of taxonomic interest (100-200bp); (4) melting curve analysis; and (5) statistical data analysis using a specific HRM software. This chapter presents an overview of HRM analysis and application, followed by the detailed description of all the required reagents, instruments, and protocols for the successful and easy implementation of a HRM method to differentiate closely related plant species.