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首页> 外文期刊>Journal of chromatography, A: Including electrophoresis and other separation methods >Fast and sensitive detection of genetically modified yeasts in wine
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Fast and sensitive detection of genetically modified yeasts in wine

机译：快速，灵敏地检测葡萄酒中的转基因酵母

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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.
机译：在这项工作中，开发了一种基于多重聚合酶链反应（PCR）和毛细管凝胶电泳与激光诱导荧光（CGE-LIF）结合使用的新型筛选方法，用于快速，灵敏地检测葡萄酒中的转基因酵母。作为模型，选择了重组EKD-13酿酒酵母菌株，并使用重组酵母或常规酵母制备了不同的葡萄酒。为了克服从葡萄酒样品中获得可扩增DNA的重要困难，特别强调酵母DNA提取步骤，探索不同的商业和非商业方法。为了明确检测转基因酵母，扩增了转基因构建体的两个特定区段。另外，将第三对引物用作扩增对照以确认从提取步骤获得的酵母DNA的质量。 CGE-LIF具有很高的灵敏度，良好的分析速度和令人印象深刻的DNA片段分辨率，从而使该技术非常方便地优化多重PCR参数和分析扩增的DNA片段。因此，CGE-LIF方法在相同毛细管条件下，DNA迁移时间的％RSD值低于0.82％（n = 10），而在三种不同毛细管条件下，DNA迁移时间低于1.92％（n = 15），因此可以确定合适的大小。与理论值相比，PCR产物的误差低于4％。这项工作中开发的整个方法需要少于一个工作日，并且可以对葡萄酒样品中的转基因酵母进行灵敏的检测。

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