摘要
【目的】为了确保转基因检测技术的标准化和准确性,开发一种适用于转基因大豆定性定量检测的多靶标质粒标准分子。【方法】针对13种转基因大豆转化体的分子特征信息,合成了含有多靶标序列的核酸片段,通过将外源片段插入pUC57质粒多克隆位点并经大肠杆菌转化,研制了含有27个转基因大豆靶标序列的质粒DNA标准分子pUC57-SOY。采用普通PCR、单重、双重实时荧光定量PCR(qPCR)检测方法对DNA标准分子pUC57-SOY的靶标特异性进行测试,利用13种大豆转化体进行定量检测适用范围和定值参数性能进行评估。【结果】在普通PCR检测中,质粒DNA包含的24种检测靶标,均能稳定扩增,且无非特异性扩增产物,表明质粒DNA标准分子pUC57-SOY在实际检测中具有良好的特异性;实时荧光定量PCR(qPCR)方法测试表明,质粒标准分子的不同浓度与靶标扩增效率具有良好的线性相关性。以pUC57-SOY标准分子为阳性参照物,应用标准曲线内插法,实际样品检测结果与预期相一致。【结论】开发的多靶标质粒标准分子pUC57-SOY表现出符合转基因成分定性定量检测要求的优良性能。在定性检测中,24种检测靶标均能稳定检测出并具有良好的特异性;在定量检测中,可实现一个标准分子对应13种靶标的精准测定。因此,该研究创制的质粒标准分子pUC57-SOY可作为转基因大豆检测的阳性对照品。
【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
作者
刘奕君
闫伟
何禹璇
董立明
龙丽坤
李飞武
LIU Yi-jun;YAN Wei;HE Yu-xuan;DONG Li-ming;LONG Li-kun;LI Fei-wu(Jilin Academy of Agricultural Sciences,(Northeast Agricultural Research Center of China),Changchun 130033;College of Life Science,Jilin Agricultural University,Changchun 130061)
出处
《生物技术通报》
CAS
CSCD
北大核心
2024年第11期169-183,共15页
Biotechnology Bulletin
基金
吉林省科技发展计划(20220505025ZP)
吉林省农业科技创新工程项目(CXGC2023RCY018)。
关键词
转基因成分检测
转基因大豆
质粒DNA标准分子
定量
detection of transgenic composition
transgenic soybean
plasmid DNA standard molecule
quantifative
