转基因玉米59122品系的特异性检测

Event-specific Transgenic Detection of Genetically Modified Maize 59122 with Flanking Sequence

使用反向聚合酶链式反应(PCR)技术克隆了转基因玉米59122的外源基因与玉米基因组之间的两段侧翼序列,并据其左侧侧翼序列设计了具品系特异性的引物,运用半巢式PCR技术建立了59122的品系特异性二重PCR检测方法,扩增片段100bp,横跨pat终止子与转基因玉米侧翼基因之间。以转基因玉米59122、MON863、MON810、GA21、NK603,转基因大豆Roundup Ready和转基因油菜GT73等为材料,证明本方法与其他转基因作物具有高特异性。本方法在检测59122时,确定出连接体系中线性DNA的最佳质量浓度为1ng/μL左右,检出限达到0.1%,灵敏度为38个单倍体基因组拷贝数。因此可准确、快速、高效地检测转基因玉米及其产品,或作为常规PCR定性检测后的验证方法。

We report the cloning of two flanking sequence of the integrated gene construct of genetically modified maize 59122 by inverse PCR method and the design of even-specific primers based on the left flanking sequence with the aim of developing of a duplex PCR assay for the event-specific transgenic detection of genetically modified maize 59122 using semi-nested PCR,result ing in an amplification fragment of 100 bp in length stretching from the terminator of the pat gene to the 59122 flanking genes. This assay has been successfully applied to detect genetically modified maizes 59122,MON863,MON810,GA21,NK603,genetically modified Roundup Ready soybeans and genetically modified oilseed rape GT73,with high specificity. The optimum concentration of linear DNA in a connection system for detecting genetically modified maize 59122 by this assay was around 1 ng/μL,which exhibited a limit of detection of 0.1% and a sensitivity of 38 copies of haploid genome. Therefore,the developed PCR assay is applicable to detect genetically modified maize and its derivates accurately,fast and efficiently,and can serve to verify routine PCR qualitative detection.