应用单管巢式和半巢式PCR检测转基因玉米MON89034
建立转基因玉米MON89034的转化体特异性检测方法,是依据转基因食品标识管理制度对其进行监管的重要技术基础。本研究根据MON89034玉米的5’端和3’端边界序列分别设计了1组转化体特异性的巢式PCR引物,并采用中途进退式PCR策略建立了MON89034玉米的转化体特异性检测方法,扩增产物分别为491 bp和188 bp。以转基因玉米MON89034及8种其他转基因作物为材料,证明本方法对MON89034玉米具有高度特异性,灵敏度测试结果表明本方法的相对检出限达到0.01%,绝对检出限为4个单倍体基因组拷贝数,比普通PCR提高了5倍。本研究建立的单管巢式和半巢式PCR方法可准确、高效地检测转基因玉米MON89034及其产品。
英文摘要:
The establishment of event-specific detection method for genetically modified (GM) maize MON89034 was the important technical assistance for the efficiently execution of genetically modified organisms labeling policies. The two sets of event-specific nested PCR primers were designed based on the 5’- and 3’- flanking sequence of the exogenous integrant of GM maize MON89034 respectively, with the aim of developing a drop-in drop-out PCR assay for the event-specific detection of GM maize MON89034, resulting in two amplification fragment of 491 bp and 188 bp in length respectively. This assay has been successfully applied to distinguished GM maize MON89034 from GM maize MON810, MON863, Bt11, Bt176, MON88017, GM Roundup Ready soybean and GM canola GT73 with high specificity. Sensitivity test results showed that the relative limit of detection of this method reaches 0.01%, equivalent to the absolute sensitivity of 4 copies of haploid genome, which was 5 times more than conventional PCR. Consequently, the single tube nested and semi-nested PCR assay established in this study can be used to accurately and efficiently detect GM maize MON89034 and its processed products.
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