Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested.

中文翻译：

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使用数字液滴 PCR 鉴定最常见转基因作物的单目标分类单元特异性参考测定


了解特定分类单元参考检测所针对的 DNA 序列数量对于正确的 GM 定量至关重要，也是协调测量结果的关键。在本研究中，液滴数字 PCR (ddPCR) 用于确定分类单元特异性检测的 DNA 靶拷贝数，该检测经过验证可用于四种主要转基因 (GM) 作物的实时 PCR。还探讨了实验条件从实时 PCR 到 ddPCR 的可转移性，以及 DNA 消化的影响。这项研究的结果表明，对于每种作物，至少有一种分类单元特异性测定可以被鉴定为具有单一 DNA 靶标。提出了一份简短的分类单元特异性参考测定清单，作为大豆、玉米、棉花和油菜转基因事件相对定量的最佳候选。在大多数情况下，所研究的测定可以转移到 ddPCR，无需进一步优化。在测试条件下，使用 DNA 消化并没有改善 ddPCR 特性，例如降雨和分辨率。