title: Fast and sensitive detection of genetically modified yeasts in wine.
abstract: In this work, a novel screening methodology based on the combined use of multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) is developed for the fast and sensitive detection of genetically modified yeasts in wine. As model, a recombinant EKD-13 Saccaromyces cerevisiae strain was selected and different wines were prepared using either recombinant or conventional yeasts. Special emphasis is put on the yeast DNA extraction step, exploring different commercial and non-commercial methods, in order to overcome the important difficulty of obtaining amplifiable DNA from wine samples. To unequivocally detect the transgenic yeast, two specific segments of the transgenic construction were amplified. In addition, a third primer pair was used as amplification control to confirm the quality of the yeast DNA obtained from the extraction step. CGE-LIF provides high sensitivity, good analysis speed and impressive resolution of DNA fragments, making this technique very convenient to optimize multiplex PCR parameters and to analyze the amplified DNA fragments. Thus, the CGE-LIF method provided %RSD values for DNA migration times lower than 0.82% (n=10) with the same capillary and lower than 1.92% (n=15) with three different capillaries, allowing the adequate size determination of the PCR products with an error lower than 4% compared to the theoretically expected. The whole method developed in this work requires less than one working day and grants the sensitive detection of transgenic yeasts in wine samples.
authors.affiliation: Laboratory of Foodomics, Institute of Food Science Research (CSIC), Nicolas Cabrera 9, Campus de Cantoblanco, 28049 Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: León
authors.forename: Virginia
authors.initials: V
authors.lastname: García-Cañas
authors.forename: Ramón
authors.initials: R
authors.lastname: González
authors.forename: Pilar
authors.initials: P
authors.lastname: Morales
authors.forename: Alejandro
authors.initials: A
authors.lastname: Cifuentes
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D030781:Organisms, Genetically Modified / Q000235:genetics*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014920:Wine / Q000382:microbiology*