玉米转基因成分三重实时荧光PCR筛选检测

Triple Real-time Fluorescent PCR Screening of Transgenic Components in Maize

本研究开发建立了三重实时荧光PCR反应体系对玉米及其加工制品的转基因成分进行高通量快速检测。以玉米单拷贝内源基因adh1(编码乙醇脱氢酶的基因)、启动子(35S promoter from cauliflower mosaic virus, p-35S)和t-NOS终止子(terminator of nopaline synthase gene from Agrobacterium tumefaciens, t-NOS)等常用转基因元件为靶标基因,以转基因玉米MON863、NK603、MON810、Bt11,非转基因玉米粉2019054以及转基因大豆粉,棉花籽和小麦粉等非玉米农作物样本共8份测试样品进行方法特异性分析,对10个玉米品系种子样本和41份玉米食品样品进行方法适用性分析和日常样本筛查检测,用标准曲线法验证方法的检测低限和重复性。结果表明该体系能在转基因阳性样本DNA为模板的一个反应管中同时检出3个靶标基因,在非转基因的玉米样本中仅检出内源基因,检测结果符合标示值;adh1、p-35S和t-NOS三种靶标基因扩增效率分别为96.84%、94.92%和93.80%,线性相关系数分别为0.999、0.990和0.997,检测低限为10个拷贝。该方法的建立可用于玉米等同源产品的出入境检测以及市场流通的符合性筛查检测。

In this study, a triple real-time fluorescent PCR system was developed for rapid detection of genetically modified components in maize and processed products. Specific primers and probes were designed for single copy endogenous gene adh1(encoding alcohol dehydrogenase), promoter(p-35S promoter of cauliflower mosaic virus) and t-NOS terminator(gene terminator of carmine synthetase of Agrobacterium tumefaciens). Specificity analysis was performed on 8 samples of transgenic maize MON863, NK603, MON810, Bt11, non-transgenic corn meal 2019054 and transgenic soybean flour, cotton seed and wheat flour applicability analysis and routine sample screening detection were performed on 10 maize seed samples and 41 maize food samples. Standard curve method was used to verify the detection limit and repeatability of the method. The results showed that the system could simultaneously detect 3 target genes in a reaction tube using transgenic positive sample DNA as template, and only endogenous gene was detected in non transgenic maize samples, and the detection results were consistent with the information of the samples to be detected. The amplification efficiency of adh1, p-35S and t-NOS target genes were96.84%, 94.92% and 93.80% respectively. Their linear correlation coefficients were 0.999, 0.990 and 0.997 respectively. The detection limit was 10 copies each. This method can be used for entry-exit inspection of maize and other homologous products as well as for market circulation compliance screening.