Abstract:

【Objective】 In order to ensure the standardization and accuracy of transgenic detection technology, this study is aimed to develop a multi-target plasmid standard molecule suitable for qualitative and quantitative detection of transgenic soybean. 【Method】 In order to characterize molecular features of 13 transgenic soybean events, nucleic acid fragments containing multi-target sequences were synthesized. These fragments were inserted into the multiple cloning sites of the pUC57 plasmid and transformed into Escherichia coli, resulting in the development of a plasmid DNA standard molecule, pUC57-SOY, containing 27 transgenic soybean target sequences. The target specificity of the DNA standard molecule pUC57-SOY was tested using conventional PCR, single, and duplex real-time fluorescence quantitative PCR（qPCR）detection methods. The applicability range and quantitative parameters were evaluated using 13 transgenic soybean events for quantitative detection. 【Result】 In PCR detection, all 24 target sequences contained in the plasmid DNA were consistently amplified without non-specific amplification products, indicating the excellent specificity of the plasmid DNA standard molecule pUC57-SOY in practical detection. Real-time fluorescence quantitative PCR（qPCR）analysis demonstrated a good linear correlation between different concentrations of the plasmid standard molecule and target amplification efficiency. Using pUC57-SOY as a positive reference, the actual sample detection results were consistent with expectations when applying the standard curve interpolation method. 【Conclusion】 The multi-target plasmid standard molecule pUC57-SOY developed in this study showed excellent performance in line with the requirements of qualitative and quantitative detection of transgenic components. In qualitative detection, 24 detection targets can be stably detected and have good specificity. In quantitative detection, a standard substance corresponding to 13 kinds of targets can be accurately determined. Therefore, the plasmid standard molecule pUC57-SOY created in this study can be used as a positive control product for the detection of transgenic soybeans.

Key words: detection of transgenic composition, transgenic soybean, plasmid DNA standard molecule, quantifative