转基因甜菜品系H7-1的数字PCR定量检测方法

Method for Quantitative Determination of Genetically Modified Sugar Beet Line H7-1 by Using Digital Polymerase Chain Reaction

摘要:

为实现转基因甜菜品系H7-1的标识管理和精准定量，根据H7-1的5’边界序列和甜菜谷氨酰胺合成酶基因（glutamine synthetase，GS）设计引物和探针建立双重数字PCR检测体系。该方法的特异性、灵敏度、精密度和准确度均进行了测试。结果显示：建立的转基因甜菜H7-1数字PCR检测方法特异于H7-1品系检测，在20 μL反应体中H7-1品系特异性序列和内源基因GS的定量下限（limit of quantitation，LOQ）分别为3.1拷贝/μL和6.3拷贝/μL，检测下限（limit of detection，LOD）检出限分别为0.6拷贝/μL和1.3拷贝/μL，精密度和准确度在可接受范围内，该定量方法不依赖于标准曲线建立，可便捷的应用于转基因甜菜H7-1成分的精确定量检测。

Abstract:

In order to achieve labeling management and accurate quantification of genetically modified sugar beet H7-1, primer pairs and probes based on the 5? flanking sequence and glutamine synthetase (GS) of H7-1 were designed, and a duplex digital polymerase chain reaction (dPCR) detection method for H7-1 was established. The specificity, sensitivity, precision, and accuracy of the developed method were examined. The results showed that the developed dPCR method was specific for line H7-1 detection. The limits of quantitation (LOQs) of the specific sequence and endogenous GS gene of line H7-1 in a 20-μL reaction system were 3.1 copies/μL and 6.3 copies/μL respectively. The limits of detection (LODs) of the specific sequence and endogenous GS gene of line H7-1 in a 20 μL reaction system were 0.6 copies/μL and 1.3 copies/μL respectively. The precision and accuracy were all in the acceptable range. This quantitative method does not rely on the establishment of a standard curve, and can be easily applied for accurate quantification of sugar beet H7-1.