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

中文关键词:  转基因马铃薯  双重数字PCR  定量检测

英文关键词:genetically modified potato  duplex digital polymerase chain reaction  quantitative detection

中文摘要:

      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r2>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。

英文摘要:

      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 r2 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.