Owing to the economic value of its foliage, tobacco (Nicotiana tabacum) is cultivated all across the world. For the detection of genetically modified (GM) tobacco, there is a lack of universal standard material which ultimately limits the detection methods because the accuracy and comparability of the results cannot be ensured. Here, we prepared a reference plasmid “pGMT27” for the detection of GM tobacco, which was 18,296 bp in length harboring two of the tobacco endogenous and seven exogenous genes. By using qualitative PCR test for the nine genes, 10 copies were used for plasmid sensitivity. In the quantitative real-time PCR (qPCR) assays with pGMT27 as a calibrator, the reaction efficiencies for P-35S and NR were 101.427% and 98.036%, respectively, whereas the limit of detection (LOD) and limit of quantification (LOQ) were 5 copies and 10 copies per reaction. For standard deviation (SD) and relative standard deviation (RSD) of the Ct values, the repeatability values were from 0.04 to 0.42 and from 0.18% to 1.29%, respectively; and the reproducibility values were from 0.04 to 0.39 and from 0.18% to 1.14%, respectively. For the unknown sample test, the average conversion factor (Cf) was 0.39, and the accuracy bias was from −15.55% to 1.93%; for precision, the SD values ranged from 0.02 to 0.62, while RSD values were from 1.34% to 10.6%. We concluded that using the pGMT27 plasmid as a calibrator provided a highly efficient transgenic detection method for flue-cured tobacco.

中文翻译：

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一种新的参考质粒“pGMT27”为烤烟提供了一种高效的转基因检测方法

由于其叶子的经济价值，烟草（Nicotiana tabacum）在世界各地都有种植。对于转基因烟草的检测，由于无法保证结果的准确性和可比性，缺乏通用的标准物质，最终限制了检测方法。在这里，我们制备了一个用于检测转基因烟草的参考质粒“pGMT27”，它的长度为 18,296 bp，包含两个烟草内源基因和七个外源基因。通过对九个基因使用定性 PCR 测试，10 个拷贝用于质粒敏感性。在使用 pGMT27 作为校准品的实时定量 PCR (qPCR) 分析中，P-35S和NR的反应效率分别为 101.427% 和 98.036%，而检测限 (LOD) 和定量限 (LOQ) 分别为每个反应 5 个拷贝和 10 个拷贝。对于 Ct 值的标准偏差 (SD) 和相对标准偏差 (RSD)，重复性值分别为 0.04 至 0.42 和 0.18% 至 1.29%；重现性值分别为 0.04 至 0.39 和 0.18% 至 1.14%。对于未知样本测试，平均转换因子（Cf）为0.39，准确度偏差为-15.55%至1.93%；对于精密度，SD 值范围为 0.02 至 0.62，而 RSD 值范围为 1.34% 至 10.6%。我们得出结论，使用 pGMT27 质粒作为校准品为烤烟提供了一种高效的转基因检测方法。