SHORT COMMUNICATION A culture-independent PCR-based method for the detection of Lachancea thermotolerans in wine Giacomo Zara & Maurizio Ciani & Paola Domizio & Severino Zara & Marilena Budroni & Adriana Carboni & Ilaria Mannazzu Received: 13 December 2012 / Accepted: 2 April 2013 / Published online: 25 April 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract When inoculated in association with Saccharo- myces cerevisiae, the yeast Lachancea thermotolerans de- termines a reduction of volatile acidity and an increase in the production of glycerol, 2-phenylethanol, and polysaccha- rides. Moreover, L. thermotolerans is a natural L-lactic acid producer, thus it contributes to wine acidification and mi- crobiological stabilization. In view of its utilization in winemaking, a culture-independent PCR-based method was developed for the detection of L. thermotolerans during wine fermentations. This method, which utilizes species- specific PCR primer pairs that anneal to intron 2 of the mitochondrial COX1 gene, is rapid and reliable, and detects L. thermotolerans in wine at 10 4 cells/ml and with a S. cerevisiae/L. termotholerans ratio of 1,000/1. Key words Lachancea thermotolerans . Wine . Mixed starters . COX1 Findings Non-Saccharomyces yeasts are metabolically active during must fermentations and contribute to the definition of wine aroma and complexity. For these reasons, several authors have proposed their utilization in mixed cultures with Saccharomy- ces cerevisiae for the inoculation of grape must. The association made by S. cerevisiae and Lachancea thermotolerans was recently described as particularly promising (Comitini et al. 2011) as it results in the reduction of volatile acidity and in the increase of glycerol, 2-phenyl-ethanol, and polysaccharides. Moreover, L. thermotolerans is a natural L-lactic acid producer with interesting potential in the acidification of wine. Recently, L. thermotolerans was utilized in mixed starters with S. cerevisiae for wine-making at the industrial scale (Gobbi et al. 2013). In this case, the persistence of L. thermotolerans during must fermentation was evaluated by viable plate counts and required up to 5 days. Here, with the aim of contributing to the development of a rapid and reliable technique for the detection of L. thermotolerans during must fermentation, a culture- independent PCR-based method was developed. To do that, the attention was focussed on mitochondrial COX1 gene coding for the subunit 1 of cytochrome C oxidase. This gene had already been considered as the molecular target of choice for S. cerevisiae detection during wine fermentations (Lopez et al. 2003) and more in general as fungal marker (Seifert et al. 2007; Mouhamadou et al. 2008; Molitor et al. 2010) due to its high inter- and intra-specific variability (Foury et al. 1998; Lopez et al. 2003). Lachancea thermotolerans COX1 gene contains three introns measuring 1,530, 1,398, and 1,404 bp (Talla et al. 2005). BLAST analyses of the three introns indicated that introns 1 and 3 show significant homologies with those of the corresponding gene of other species of oenological interest, therefore they cannot be considered targets of interest for L. thermotolerans molecular mon- itoring. In contrast, intron 2 did not show homologues in the database. Thus, by utilizing Primer 3 software, primers INT2F (5′-TGGTTTTATTGAAGCCAAAGG-3′) G. Zara : S. Zara : M. Budroni : A. Carboni : I. Mannazzu (*) Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy e-mail: imannazzu@uniss.it M. Ciani Dipartimento di Scienze della Vita e dell’Ambiente (DiSVA), Università Politecnica delle Marche, Via Brecce Bianche, 60100 Ancona, Italy P. Domizio Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Via Donizetti 6, 50144 Firenze, Italy Ann Microbiol (2014) 64:403–406 DOI 10.1007/s13213-013-0647-4