Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction
Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction
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DOI：
10.1007/s00216-009-3200-3
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发表时间：
2010-03-01
影响因子：
4.3
通讯作者：
Emslie, Kerry R.
中科院分区：
文献类型：
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作者：
Corbisier, Philippe;Bhat, Somanath;Emslie, Kerry R.
转基因食品的定量分析需要估计转基因事件相对于内源基因的量。欧洲联盟（欧盟）的规管当局已根据转基因事件与内源基因的复制数比率，界定基因改造食物的标签门槛。实时聚合酶链反应（PCR）目前正用于定量转基因生物（GMO）。在实时PCR应用中检测非常低数量的DNA靶标的限制导致了新的发展，例如数字PCR（dPCR），其允许在不需要参考校准物的情况下精确测量DNA拷贝。在本文中，玉米MON810和hmg拷贝的数量存在于DNA提取物中的种子粉末认证其质量含量和拷贝数比通过dPCR测量。发现通过dPCR测定的这些绝对拷贝数的比率与使用质粒DNA校准品通过实时定量PCR（qPCR）测量的比率相同。这些结果表明，这两种方法都可以用于确定MON810中的拷贝数比。报告的值与从模型中的估计值一致，该模型是为了将质量分数转换为MON810品种的拷贝数分数而精心设计的。该模型在用于生产MON810认证参考物质（CRM）的两个MON810品种上受到挑战，这两个品种在引入的转基因性状的亲本来源上不同。我们的结论是，dPCR具有较高的检测质量，可用于证明转基因标准物质的DNA拷贝数比。
Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.