Zhang David, Corlet Aurélie, Fouilloux Stephane
GEVES Domaine du Magneraud, Laboratoire BioGEVES, B.P. 52, Surgeres 17700, France.
Transgenic Res. 2008 Jun;17(3):393-402. doi: 10.1007/s11248-007-9114-y. Epub 2007 Jul 19.
Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% x (0.5 +/- 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N x GM wt% x (0.5 +/- 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.
基于实时聚合酶链反应（PCR）的检测方法被广泛用于估计食品、饲料和种子中转基因（GM）材料的含量。众所周知，分析物的遗传结构会显著影响通过实时PCR检测估计的单倍体基因组（HG）%所表示的转基因含量；这种影响也被理解为生物因素的影响。这种影响首先在理论层面以玉米为模型进行了模拟。然后，我们通过定量实时PCR分析了六个具有不同遗传结构的玉米MON 810杂交籽粒，实验评估了生物因素对定量结果的影响：（1）转基因雄性亲本的半合子，（2）转基因雌性亲本的半合子，以及（3）转基因位点的纯合子。本研究获得的结果表明生物因素对转基因DNA定量有明显影响：从定量实时PCR分析来看，三种遗传结构中按重量计1%的转基因材料分别含有0.39%、0.55%和1.0%的转基因DNA（按HG计）。转基因重量%与转基因HG%之间的关系可以通过经验确定为：（1）在存在单个转基因性状的情况下：转基因HG% = 转基因重量% x （0.5 +/- 0.167Y），其中Y是玉米籽粒总DNA中的胚乳DNA含量（%），（2）在存在多个转基因性状的情况下：转基因HG% = N x 转基因重量% x （0.5 +/- 0.167Y），其中N是未知样品中存在的转基因性状（堆叠与否）的数量。这一发现可供转基因相关利益相关者在种子和谷物生产链监测中从一种表达单位到另一种表达单位进行经验预测时使用。还提出了用于单倍体拷贝数计算的实用公式，使用半合子转基因材料制作校准曲线。
