Development and Validation of an Event-Specific Quantitative PCR Method for Genetically Modified Maize MIR162
Development and Validation of an Event-Specific Quantitative PCR Method for Genetically Modified Maize MIR162
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DOI：
10.3358/shokueishi.55.205
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发表时间：
2014-10-01
影响因子：
0.3
通讯作者：
Kitta, Kazumi
中科院分区：
文献类型：
--
作者：
Takabatake, Reona;Masubuchi, Tomoko;Kitta, Kazumi
建立了一种新的基于实时PCR的分析方法，用于转基因玉米事件MIR162的事件特异性定量。我们首先制备了用于MIR162定量的标准质粒。对两台实时聚合酶链式反应仪器--应用生物系统7900HT(ABI7900)和应用生物系统7500(ABI7500)--计算转基因生物量所需的换算因子(Cf)进行了经验测定，其Cf值分别为0.697和0.635。为了验证所开发的方法，在实验室间研究中进行了盲法测试。以相对标准偏差(RSDR)的偏倚和重现性为评价方法的真实性和精密度。在所有评价浓度下，测定的偏差均小于25%，RSDR值均小于20%。结果表明，该方法的定量限为0.5%，适合于MIR162的检测和定量分析。
A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize event, MIR162. We first prepared a standard plasmid for MIR162 quantification. The conversion factor (Cf) required to calculate the genetically modified organism (GMO) amount was empirically determined for two real-time PCR instruments, the Applied Biosystems 7900HT (ABI7900) and the Applied Biosystems 7500 (ABI7500) for which the determined Cf values were 0.697 and 0.635, respectively. To validate the developed method, a blind test was carried out in: an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDR). The determined biases were less than 25% and the RSDR values were less than 20% at all evaluated concentrations. These results suggested that the limit of quantitation of the method was 0.5%, and that the developed method would thus be suitable for practical analyses for the detection and quantification of MIR162.