Detection of wine spoilage microorganisms by DNA microarray assay

The goal of this study has been the design and the validation of an oligonucleotide microarray in order to detect 17 different wine-spoilage microorganisms, i.e. 9 yeast, 5 lactic bacteria and 3 acetic acid bacteria species. Oligonucleotide probes specific to each microorganism were designed to target the intergenic spacer regions (ISR) between18S-5.8S region for yeasts and 16S-ITS1 region for bacteria. Before hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consensus primer. Each oligonucleotide-probe solely recognized its target without undesired aspecific cross-hybridizations. Under our experimental condition, the microarray assay analysis was able to detect the amount of DNA equivalent to 24 (Saccharomyces cerevisiae), 160 (Lactobacillus brevis) and 124 (Gluconobacter oxydans) cells, three microbes chosen as experimental models. Furthermore, a new procedure that allowed the extraction of genomic DNA from a mixture of eukaryotic and prokaryotic cells from contaminated wine was developed. The microarray methodology has been applied for the first time to simultaneously identify different mixed population of spoilage yeast and bacteria directly from wine. The significance and the impact of the above findings will be discussed. This research was partially supported by the Apulian Region Projects "Biotecnologie degli alimenti per l'innovazione e la competitività delle principali filiere regionali: estensione della conservabilità e aspetti funzionali - BIOTECA -QCBRAJ6." and "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)".