实时荧光定量PCR(qRT-PCR)检测转基因成分的数据分析及其标准化研究  

Standardization of Data Analysis in Real-time Quantitative PCR Detection of Genetically Modified Ingredients

作　　者：张丽[1,2] 曹应龙[2] 王海英[1] 梁晓声[1] 卢长明[2] 

机构地区：[1]中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北省重点实验室,武汉430074 [2]中国农业科学院油料作物研究所/农业部油料作物生物学重点开放实验室,武汉430062

出　　处：《农业生物技术学报》2015年第1期126-134,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金资助项目(No.31401607);转基因生物新品种培育重大专项(No.2013ZX08012-005);环保公益性行业专项(No.201109028)

摘　　要：在利用实时荧光定量PCR(Real-time quantitative PCR,q RT-PCR)检测转基因成分的含量时,数据分析的规范化与标准化对保证实验数据的准确性及实验室间数据的可比性具有重要意义。本研究以转基因玉米(Zea mays)NK603为研究材料,分析了分别以q RT-PCR中3次平行反应的单个Ct值和3次平行反应Ct值的平均值所绘制标准曲线的差异;分析了分别以盲样3次平行反应的Ct平均值和3次平行反应的拷贝数平均值(算术平均值或几何平均值)计算转基因成分含量的差异;并研究了结果准确性判定的方法。结果表明,标准曲线应基于单个Ct值;在计算转基因成分的含量时,Ct值应取算术平均值,拷贝数应取几何平均值;最后通过比较样品的测量结果与标准值之间的差异是否显著(UΔ>Δm,表明测量结果的平均值与样品的标准值无显著差别)来判断实验结果的可靠性。本研究为转基因产品检测中q RT-PCR实验数据处理的标准化提供了参考资料。In using Real-time quantitative PCR （qRT-PCR） detection of genetically modified （GM） ingredients, the standardization of data analysis is of great significance for the accuracy of the experimental data and the comparability of inter-laboratory data. This research was designed to promote the standardization of data analysis in GM ingredients quantitative detection. Taking the GM maize （Zea mays） NK603 as an example in qRT-PCR analysis, maize endogenous reference gene alcohol dehydrogenase 1 （Adhl） and NK603 event-specific sequence were set as amplified targets. Two certified reference materials （GM content of 0.98% and 4.91%, respectively） were used as blind samples and the GM content were measured. In qRT-PCR assays, standard curves were generated based on the least-square method, and GM content of blind samples was measured through the approach of absolute quantification. The construction of standard curves and analysis of blind samples were repeated 3 times, and 3 parallel reactions were included in one plate for each time.Calibration curves were produced using the mean Ct values of three parallels or using the individual Ct values of three parallels. Corresponding, the GM content of blind samples should be calculated firstly based on mean of Ct values and then transformed to copies, or calculated firstly based on individual copies which was transformed from individual Ct values and then taking the geometric mean or arithmetic mean of copies. It was considered that different treatments on data lead to varied accuracy of the results. In order to enhance the comparability of interlaboratory data, our results suggested that standard curves should be produced using the original Ct values rather than the mean of the Ct values, and the GM content of blind samples should be based on arithmetic mean of Ct values or geometric mean of copies. The accuracy of the test should be judged through comparing the measurement results with the certified value （U△〉△m, which indicating there was no signif

关 键 词：实时荧光定量PCR(qRT-PCR) 转基因成分 定量检测 标准曲线 数据分析 

分 类 号：O171[理学—数学]