title: An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR.
abstract: The availability of genetically modified (GM) yeasts for winemaking and, in particular, transgenic strains based on the integration of genetic constructs deriving from other organisms into the genome of Saccharomyces cerevisiae, has been a reality for several years. Despite this, their use is only authorized in a few countries and limited to two strains: ML01, able to convert malic acid into lactic acid during alcoholic fermentation, and ECMo01 suitable for reducing the risk of carbamate production. In this work we propose a quali-quantitative culture-independent method for the detection of GM yeast ML01 in commercial preparations of ADY (Active Dry Yeast) consisting of efficient extraction of DNA and qPCR (quantitative PCR) analysis based on event-specific assay targeting MLC (malolactic cassette), and a taxon-specific S. cerevisiae assay detecting the MRP2 gene. The ADY DNA extraction methodology has been shown to provide good purity DNA suitable for subsequent qPCR. The MLC and MRP2 qPCR assay showed characteristics of specificity, dynamic range, limit of quantification (LOQ) limit of detection (LOD), precision and trueness, which were fully compliant with international reference guidelines. The method has been shown to reliably detect 0.005% (mass/mass) of GM ML01 S. cerevisiae in commercial preparations of ADY.
authors.affiliation: CREA-Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria - Centro di Ricerca per l'Enologia, Via Pietro Micca 35, 14100 Asti, Italy. Electronic address: enricotommaso.vaudano@crea.gov.it.
authors.forename: Enrico
authors.initials: E
authors.lastname: Vaudano
authors.affiliation: CREA-Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria - Centro di Ricerca per l'Enologia, Via Pietro Micca 35, 14100 Asti, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Costantini
authors.affiliation: CREA-Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria - Centro di Ricerca per l'Enologia, Via Pietro Micca 35, 14100 Asti, Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Garcia-Moruno
mesh_terms: D005285:Fermentation; D005819:Genetic Markers / Q000235:genetics; D019344:Lactic Acid / Q000096:biosynthesis; D008291:Malate Dehydrogenase / Q000235:genetics*; D008293:Malates / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012269:Ribosomal Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000145:classification* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014920:Wine / Q000032:analysis / Q000382:microbiology*; D015002:Yeast, Dried
chemical_list: D005819:Genetic Markers; C093947:MRP2 protein, S cerevisiae; D008293:Malates; D024101:Mitochondrial Proteins; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019344:Lactic Acid; C030298:malic acid; C037731:malolactic enzyme; D008291:Malate Dehydrogenase
keywords: GMO; ML01; Saccharomyces cerevisiae; qPCR