DNA analysis in wines: Development of methods for enhanced extraction and real-time polymerase chain reaction quantification

Reliable methods for DNA traceability in grapevines and wines is in great demand for protecting areas of declared origin and detecting potential transgenic events. Currently, real-time polymerase chain reaction (PCR) is one of the most promising tools for conducting plant and microorganism DNA assays and detecting genetically modified material. However, in grape, quantitative analysis based on PCR is lagging behind. Moreover, in musts and wines, efficient DNA extraction and amplification need to be developed. In the present research, we compared several DNA extraction procedures on various grape tissues, musts and wines of the Trentino-Sudtirol Region. We chose the Vitis vinifera nine-cis-epoxycarotenoid dioxygenase2 (VvNCED2) gene as the referee standard in grape and tested for validation. Using real-time PCR, with VvNCED2 as standard referee, we successfully quantified grape DNA from grape tissues and the residual grape DNA in musts and wines. In addition, we also quantified the S. cerevisae residual DNA in wines. Extracted grape DNA quality and specificity were also verified by co-dominant simple sequence repeat (SSR) markers. SSRs proved that, even 1 year after wine fermentation, we could detect and identify grape DNA that was still suitable for analysis. Our research is the first successful report of efficient DNA extraction, amplification and real-time PCR quantification for aged wines. As a result, we could count on a suitable assay for tracing and monitoring endogenous and exogenous DNA in grape and grape products. (c) 2005 Elsevier B.V. All rights reserved.