The Research Of Flow Fluorescence Assay For Detection Of The GM Soybeans

With the rapid development of genetically modified organisms(GMOs), its safety is concerned about by more and more people. So many countries and regions are issuing about labeling regulations on GMOs and this products. Currently, huge amounts and diversity of GMOs have been destroyed after deeply processed, which not only increase the workload, but also increase the difficulty of detection. Therefore, the establishment of a multi-line, sensitive, specific, and highly reproducible of the rapid detection method is very necessary.The aim of the study was to establish a rapid molecular assay to detect five kinds of GM soybeans simultaneously by combining advanced target enrichment multiplex-polymerase chain reaction(Tem-PCR)technique with flow fluorescence assay(FFA)method, and to evaluate the reliability and application of the novel assay in practical applications. The main research is as follows:First, we turned to Gen Bank, to find the sequences of transgenic integration sequence of five soybeans.The i Cubate2.0 online software was used to calculate the exogenous gene sequence connection area of polymerase affinity index(PPI), and 12 pairs of nested primers(Fo/Ro and Fi/Ri), one pair of Super Primers(Fs and Rs) and six probes for each sequence using Primer5.0 and Beacon Designer. Each of the primers should be located in a conserved region and the 3′ end of each primer should be located at a nucleotide that begins with a 10-nucleotide sequence with a relatively high PPI. The panel of potential primers was then balanced to obtain sets with similar PPIs and to increase or decrease the length of the primers to select primers with similar TMs. The 5′ end of the primers Fi and Ri were exchanged tags(the sequence tags for priming the universal primers Fs and Rs).Secondly, we set up Tem-PCR of five kinds of GM soybeans. All these specific nested primers were contained in the reaction at low concentration, and they were not labeled. Only the superprimers were at regular PCR concentration. This method will avoid to product high concentration of labeled primers form dimmers, yield non-specific products, lead to high noise levels for the detection step.Finally, we set up FFA of five kinds of GM soybeans. The reverse superprimer was biotin labeled, and it had a even higher concentration than the forward superprimer, so that most of the PCR products were single stranded. The biotin-labeled PCR products were incorporated into the hybridized probe microspheres, and combined with SA-PE. Bio-PlexTM 200 System was used to read median fluorescence intensity and determine whether it contained GMOs.In this study, we have established a multi-line, high-throughput, high specificity and sensitivity of the rapid detection system suitable for the five GM soybeans. The method is rapid, and the time of entire experiment is within five hours. The method is sensitivity, and the detecting limitation could reach to 0.01%. This study could make the technical reserve ready for the detection of the GM soybeans and provide lessons experiences and a good testing platform for other rapid high-throughput detection of GMOs.