The Study On The Detection Of Transgenic Soybean And Oil By The Five-plex PCR

With the rapid development of transgenic technology, there were more and moregenetically modified organisms(GMO) and their products in the world. Geniticallymodified food contain new genetic materials inclouding exogenous DNA and protein.However, the safety issue arose internationally. Many countries adopt the labeled systemtowards the genetic modified food. The research aims to build a method which is accurate,rapid and reliable for detection of genetically modified food.Five-plex PCR is successfully established by targeted genes which include CaMV35Spromoter, Nos terminato, NPTⅡ, CP4-EPSPS and endogenous gene Lectin. DNA wasisolated by the improved method and then amplifed by PCR. Five genes are simultaneouslyspecifically detected. Commercial soybeans, genetically modified soybeans and oil weredetected with the five-plex PCR. The linear range of the method of detection is 0.1%.In present study, The reaction mixture consisted of 0.5μL of Taq(5 U/μL of stocksolution), 5μL of 10×PCR amplification buffer, 4μL of dNTPs(1mM each in solution),6μL of Mg2~+, 2μL of each primer(10μM stock solution) of Lectin, 1.9μL of eachprimer(10μM stock solution) of CaMV, 1.9μL of each primer(10μM stock solution) ofNPTⅡ, 2μL of each primer(10μM stock solution) of CP4-EPSPS, 1.7μL of eachprimer(10μM stock solution) of NOS, DNA templates 4μL and 20μL of double-distilledwater. The reaction was run under the following conditions: Cool start; DNApre-denaturation at 94℃for 5 min; DNA denaturation at 94℃for 40s, primer annealing at57℃for 30s, and DNA extension at 72℃for 30s for 35 cycles; the last extension wasperformed at 72℃for 6 min. The PCR products were examined by electrophoresis with2% agarose gel. The amplified product was sequenced and compared with CaMV35Spromoter, Nos terminato, NPTⅡ, CP4-EPSPS, Lectin in Genebank and found to have thehomogenicity of 100%,95%,99%,100%,99%.Furthermore, Five-plex PCR is a feasible and effective genetically modified soybeansand bean product detection method and will have broad application prospect.