Key Laboratory of Biology and genetic improvement of oil crops of the Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, 430062, Hubei, China.
Anal Bioanal Chem. 2021 Jul;413(16):4341-4351. doi: 10.1007/s00216-021-03390-9. Epub 2021 May 22.
The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.
为了实施转基因生物（GMO）标签政策，需要使用准确的定量方法来测量测试样品中的 GMO 含量。本研究以纯合 KMD 水稻基因组 DNA（gDNA）为模板，通过优化退火温度和循环数，建立了 Kemingdao/磷脂酶 D（KMD/PLD）双 ddPCR 方法。双 ddPCR 在 68 到 175000 拷贝的动态范围内呈现线性响应，涵盖了四个数量级。双 ddPCR 的检测限（LOD）和定量限（LOQ）分别为 9 个和 34 个水稻单倍体基因组拷贝。当稀释倍数过高时，测定 gDNA 溶液拷贝数的偏差和变异系数将变得不可接受，而在一个反应中使用超过 1000 个 DNA 模板可以获得更准确的定量结果。通过双 ddPCR、单 ddPCR 和 qPCR 对 5 个具有 10%、5%、1%、0.1%和 0.05%拷贝数比值的盲样 DNA 进行定量，对 3 个含有 5%、1%和 0.5%质量分数的实际基质盲样进行定量。这三种方法都在所需的精度范围内给出了可比的 GMO 含量和拷贝数，但与单 ddPCR 和 qPCR 相比，双 ddPCR 显示出更窄的不确定度区间和更高的精度。考虑到定量结果的不确定性和精度、生成液滴的时间消耗以及 ddPCR 试剂的成本，ddPCR 是一种比单 ddPCR 和 qPCR 更有吸引力和可靠的技术，可用于准确定量 GMO 含量。
