转基因大豆低密度基因芯片的检测方法研究

Study on Detection Method of Low-density Gene Chip of Transgenic Soybeans

目的建立Roundup Ready转基因大豆(简称RRS)的基因芯片检测方法,探讨其在转基因大豆检测中的应用。方法根据大豆中所转入的外源基因,选择camv35s启动子、nos终止子和cp4epsps基因,以大豆内源基因lectin基因为内参照基因设计了引物和探针,并制备了核苷酸芯片;通过多重PCR对样品核酸进行扩增和荧光标记后,将PCR产物与芯片杂交,检测大豆样品中所含的外源基因,并评价方法的灵敏度。结果检测转基因含量不同的RRS标准参考物,结果显示:camv35s启动子、nos终止子、外源基因cp4epsps检测灵敏度均可达0.45%。结论本研究所建立的基因芯片检测方法有良好的灵敏度及可重复性,有助于实现转基因大豆的高通量、高灵敏的检测。

Objective To develop the detection method of low-density gene chip to detect exogenous genes in transgenic soybeans simultaneously. Method Three exogenous DNA fragment, including camv35s promoter, nos termination and cp4 epsps gene, and endogenous gene lectin were selected as target genes, the primers and probes of which were designed and synthesized, then the probes were immobilized on the chip. Multiplex PCR was used to amplify the target sequences from the soybean sample meanwhile the Cy5-dUTP was adulterated into amplicons, which were hybridized with DNA microarray to evaluate the sensitivity. Results The results showed that the DNA chip for identifying various RRS Certified Reference Materials was highly specific and repeatable and the detection level of the exogenous genes in RRS was 0.45%. Conclusion The DNA microarray procedure might help to develop a method with high thoroughput and sensitivity in detecting transgenic soybean.