双重数字聚合酶链式反应定量检测转基因马铃薯EH92-527-1品系

Quantitative detection of genetically modified potato event EH92-527-1 by duplex digital polymerase chain reaction

目的建立双重数字聚合酶链式反应(polymerase chain reaction,PCR)精准定量检测转基因马铃薯EH92-527-1品系的方法。方法基于马铃薯内参UGPase基因和EH92-527-1品系外源插入片段旁侧序列,设计合成引物和探针,确定反应体系和反应条件,建立转基因马铃薯EH92-527-1品系双重数字PCR定量检测方法。对方法的特异性、定量检测范围、定量限、检测准确度进行评估。结果该方法特异性良好,除转基因马铃薯EH92-527-1品系外,其他物种和品系均无扩增;在线性范围内,内参基因和品系特异性基因拷贝数的相对标准偏差值介于0.86%~22.90%,线性决定系数r^(2)>0.99;品系特异性基因的定量限为3拷贝;不同浓度样品EH92-527-1品系含量测定值与真实值之间的偏差分别为1.54、4.92和–1.31。结论方法具有良好的重复性和准确度,可以用于进出口产品中转基因马铃薯EH92-527-1成分的定性定量检测。该方法的建立对于转基因马铃薯及其制品的监控监管、安全评价和风险预警具有重要意义。

Objective To establish a precise quantitative detection method for genetically modified potato event EH92-527-1 by duplex digital polymerase chain reaction(PCR).Methods Based on the endogenous UGPase gene of potato and the lateral sequence of the exogenous insertion fragment of event EH92-527-1,primers and probes were synthesized,and the reaction system and conditions were determined to establish a duplex digital PCR quantitative detection method for genetically modified potato event EH92-527-1.The specificity,quantitative detection range,limit of quantitation and detection accuracy of the method were evaluated.Results This method had good specificity,and except for the transgenic potato EH92-527-1 strain,no other species and strains had been amplified;within the linear range,the relative standard deviation values of the copy number of reference genes and event specific genes ranged from 0.86%–22.90%.The linear determination coefficient r^(2)was greater than 0.99.The limit of quantitation for event specific genes was 3 copies.The deviations between the measured values and the true values of EH92-527-1 strain content in samples with different concentrations were 1.54,4.92,and–1.31,respectively.Conclusion The method exhibits good repeatability and accuracy,and can be used for qualitative and quantitative detection of genetically modified potato event EH92-527-1 in import and export products.It is of great significance for the monitoring,supervision,safety evaluation,and risk warning of genetically modified potatoes and their products.