Novel authentication approach for coffee beans and the brewed beverage using a nuclear-based species-specific marker coupled with High Resolution Melting analysis

Abstract Coffee is a very popular and highly traded commodity that is targeted by fraudulent practices, affecting global production and economy. A common practice involves the substitution of the more expensive Arabica coffee with the less popular Robusta. Various physiochemical and DNA-based technologies have been used for coffee authentication; yet detection of adulterants in coffee still faces limitations in terms of the raw material used, the level of processing, and the sensitivity of available analytical tools. Additionally, DNA-based methodologies cannot be successfully used in the presence of strong inhibitors in brewed coffee. Herein, we designed an Internal Transcribed Spacer region 2 (ITS2)-based marker that allowed coffee authentication, even from the brewed beverage, when coupled with High Resolution Melting (HRM) analysis. The developed methodology could effectively detect differences in Arabica/Robusta admixtures, as low as 1% (v/v) DNA content. Direct species identification from the brewed beverage was enabled by the use of an inhibitor-tolerant DNA polymerase with enhanced sensitivity, without prior DNA purification. In the context of food safety, this novel approach could advance coffee authentication methods for safeguarding coffee producers and consumers.