Uncertainty in Detecting Specific Fragment of Genetically Modified Maize Event NK603 by Real-time Fluorescence Quantitative PCR

In this study, the event-specific real-time fluorescence quantitative PCR method established by Siehuan Academy of Agricultural Sciences was employed to detect the content of flanking fragment （specific fragment） in samples containing 2% genetically modified maize event NK603. The uncertainty of detection results was evaluated based on various uncertainty sources, such as PCR amplification system, data analysis and micropipette. The results showed that A-type uncertainty （ uA ）, B-type uncertainty （ uB ）, combined standard uncertainty （ uC ） and expanded uncertainty （ U95 ） were 0. 000 8,0.1301,0. 001 and 0. 002, respectively; the final detection result was 1.9% ±0.002. Thus, the main uncertainty in detecting flanking fragment of genetically modified maize event NK603 with realtime fluorescence quantitative PCR method was derived from the random effect in the experimental process.

Development of an Event-specific Quantitative PCR for Genetically Modified Maize (Zea mays) Event NK603

[ Objective ] The aim of this study was to develop a quantitative PCR detection method for genetically modified maize event NK603, so as to provide ba- sis for quantitative analysis of event NK603. [ Methods ] A quantitative PCR detection method for genetically modified maize event NK603 was developed using primers and Taqman probe designed according to the flanking sequence of event NK603, which was then adopted to detect the samples containing 2% NK603 stand- ard （with uncertain quantity of 10% ）. [ Results ] The slope of standard curve ranged between -3.6 and -3.1, and the correlation coefficient was higher than 0. 99. The amplification efficiency of this method reached 100.2%, fallen between 90% and 110%. The detected quantity of the experimental sample was 1.9%, closer to the true quantity （2%）. [ Conclusion] This quantitative PCR detection method for genetically modified maize event NK603 is very precise and can be a- dopted in routine testing analysis.

实时荧光定量PCR检测转基因玉米NK603品系特异片段的测量不确定度

应用转基因玉米NK603品系特异实时定量PCR方法,测定含量为2%的转基因玉米NK603样品中旁侧片段(品系特异片段)的含量,并从扩增反应、数据处理以及微量移液器等不确定度来源,评定了测量的不确定度。结果表明,u A=0.0008,u B=0.001,u C=0.001,U=0.002,测量结果为1.9%±0.002。NK603品系特异实时定量PCR方法检测结果的主要不确定度来自检验过程中的随机效应。

转基因玉米NK603品系特异定量PCR检测方法的建立

目的:建立转基因玉米NK603品系特异定量PCR检测方法,为转基因玉米NK603提供科学的定量检测依据。方法:根据转基因玉米NK603外源基因的旁侧序列设计引物和Taqman探针,建立转基因玉米NK603品系特异定量PCR检测方法,并采用该法检测2%含量的NK603标准品(不确定度为10%)。结果:采用构建的方法获得标准曲线斜率为-3.6^-3.1,相关系数大于0.99,扩增效率为100.2%,在90%~110%范围内;样品检测结果(1.9%)接近已知含量(2%,不确定度为10%)。结论:建立的转基因玉米NK603品系特异定量PCR检测方法的准确度较高,可在日常检验中推广应用。

转基因玉米NK603结构特异定量PCR方法的建立

根据转基因玉米NK603载体构建特异序列,设计引物和Taqman探针,建立了转基因玉米NK603载体构建特异结构定量PCR检测方法,并采用该法检测了2%含量的NK603标准品(不确定度为10%)。结果显示,采用本文构建的方法获得的标准曲线斜率,在-3.6～-3.1之间,相关系数大于0.99,扩增效率为98.1%,在90%～110%的范围内。样品检测结果(1.6%)接近已知含量(2%,不确定度为10%)。表明本文建立的转基因玉米NK603结构特异定量PCR检测方法,可以在日常检验中推广应用。

实时荧光定量PCR检测转基因玉米NK603结构特异基因的测量不确定度研究

应用四川省农业科学院分析测试中心实验室建立的转基因玉米NK603结构特异实时定量PCR方法测定含量为2%的转基因玉米NK603样品中结构特异片段的含量,并从扩增反应、数据处理以及微量移液器等不确定度来源,评定了测量的不确定度。结果表明,uA=0.0003,uB=0.001,uC=0.001,U95=0.002,测量结果为1.6%±0.002%。NK603结构特异实时定量PCR方法检测结果的主要不确定度来自检验过程中的随机效应。