Study On Detection And Identification Of Roundup Ready Soybean Using Oligonucleotide Microarray

With more and more complicated GMO plants under research and being approved, the traditional and widely used PCR detection method can no longer meet the demands to fast detect a great quantity of unknown GMO. The development of DNA chips (microarray) makes available a powerful and high throughput detection and screening method, which is thousands and hundreds times more efficient than the traditional one. However, the research of applying microarray technology to GMO detection is still at the primary stage.In the present study, the Roundup Ready soybean, one of the model GMO approved and with clear transgenic information, was chosen as main research material and, Btl76 and Bt11maize as supplementary research materials, the oligonucleotide microarray was used in the GMO detection and identification research, and the following key points and problems were studied and discussed.I. 15 genes including screening gene, specific gene, species specific gene, construct specific gene, event specific gene for Roundup Ready soybean gene box and other GMO contrast material were chosen, every target fragment was amplified under GMO template DNA and non-GMO template DNA. The results showed that the DNA quality extracted from the CTAB methods accorded with the demands of PCR amplification, and every target amplification was the same as expected whether it was GMO template DNA or non-GMO template DNA. It can be concluded that the PCR system worked smoothly and the template DNA was not contaminated. The direct sequencing results did not differ from the designed target amplification sequence.II. An oligonucleotide microarray was made to detect and identify Roundup Ready soybean. It was shown that the sequencing results included probe sequence or complementary sequence, and the valid signal produced was the same as expected with no difference from the electrophoresis results. The specificity of oligonucleotide microarray was good.The sensitivity and the specificity of oligonucleotide microarray are better than that of the electrophoresis method.III. The two commonly used fluorescence labeled methods: the random primer labeling and the PCR reaction labeling, were compared and studied. Judged from specificity, signal intensity and signal-to-noise index, it was generally estimated that oligonucleotide microarray of GMO detection combined with the PCR reaction labeling was optimal.IV. Two sets of multiplex PCR system to detect and identify Roundup Ready soybean were set up and optimized (4 X and 5 X multiplex PCR system respectively). Eight different genes could be detected in one oligonucleotide microarray by mixing two sets of multiplex PCR products and the goal of detecting and identifying Roundup Ready soybean could be achieved.V. 5 X multiplex PCR system for distinguishing BT176 , Btl 1 maize and non-GM maize was set up and optimized. It was shown that the three different types of maize could be effectively detected and distinguished. And it was estimated that the specificity of the oligonucleotide microarray was good.VI. Some factors affecting multiplex PCR products hybridization signals were optimized and compared with the widely used hybridization system. The result showed that the optimal hybridization time should be 40 minutes and SSC concentration should be 5 X SSC, with the other factors being the same as the condition of the widely used system. Denaturalization of labeled PCR products should be emphasized before hybridization.The results of the present study has made some helpful exploration and research in applying oligonucleotide microarray to GMO detection and has laid the groundwork for making higher density oligonucleotide microarray to detect and identify more GMO.