转基因植物中标记基因定性PCR检测方法研究

Development of qualitative PCR method targeting marker genes in transgenic plants

为了建立以标记基因为检测靶标的高效定性检测方法,本研究系统地分析了目前申请商业化的转基因植物中标记基因的应用频率,选取了应用频率高或在未来应用潜力大的标记基因neomycin phosphotransferaseII(NPTII)、phosphinothricin acetyltransferase(PAT)、5-enolpyrul-shikimate-3-phosphate synthase(CP4-EPSPS)、phosphinothricin acetyltransferase(bar)、hygromycin phosphotransferase II(HPTII)、β-glucuronidase(GUS)和phospho-mannose isomerase(PMI)作为检测靶标,建立了这七个基因的普通PCR和实时荧光PCR检测方法。该研究建立的普通PCR方法和实时荧光PCR方法特异性强、灵敏度高、重现性好,适用于农产品中转基因成分的大规模筛查检测,而且应用实时荧光PCR方法可大大提高检测效率,降低检测过程中样品交叉污染和环境污染的几率。

To establish an effective screening method targeting marker genes for detecting genetically modified plants, we systematically analyzed the current application frequency of marker genes in commercial transgenic plants, and found that seven genes, including NPTII, PAT, CP4 - EPSPS, bar, HPTII, GUS and PMI, had been used in high frequency currently or could have potential applications in the future. The conventional PCR and real -time PCR detection methods were developed with these seven genes as detecting targets. The PCR method was specific, sensitive, reproducible and suitable for large - scale screening detection of genetically modified agricultur- al products. Moreover, the application of real - time PCR detection method could be more efficient, and reduce cross - contamination of samples and risk of environmental pollution during testing procedure.