Multiplex PCR Detection Technology Of Genetically Modified Rice And Canola

With the increasing number and variety of genetically modified organisms(GMOs)and their products worldwide,the potential risks resulting from GMOs have aroused worldwide concern.The safety supervision of GMOs is closely related to food security and ecological security.The countries and regions represented by the European Union have introduced a series of laws and regulations to strengthen supervision.Detections of GMOs are important technical supports and means for the safety supervision of GMOs.In recent years,detection technologies only based on traditional Polymerase Chain Reaction(PCR)counld not satify the requirement of modern detection technology of GM components.Multiplex PCR has been widely used in the field of GM component detection due to the advantage of stronger specificity,higher sensitivity,lower cost,higher flux,and saving experimental samples and drugs.In this paper,GM rices TT51-1,KMD1 and KF6 and GM canolas RF1,MS8,Topasl9/2,Oxy235,T45,RF2,RF3 and GT73 were studied,the multiplex PCR detection technologys were developed to meet current detection and supervision demands.The main contents and results were as follows:(1)Event specific multiplex qualitative PCR detection technique for GM rice lines:Specific primers were designed for the flanking sequences of GM rice lines TT51-1,KMD1 and KF6.Multiplex qualitative PCR for GM rice lines was established.The specificity of selected primers was tested by amplifying different crops such as GM rice,GM soybean,GM maize,GM canola,and GM cotton.The results showed that the established detection method of multiplex qualitative PCR for GM rices had stronger specificity,and the limit of detection(LOD)was 0.1%.(2)Event specific multiplex qualitative PCR detection technique for GM canolas:Specific primers were designed for the flanking sequences of GM canola RF1,MS8,Topasl9/2,Oxy235,T45,RF2,RF3,GT73 lines.Multiplex qualitative PCR for GM canolas was established by using primer selection,multiplex PCR reaction system and reaction condition optimization.The selected primer specificity was tested by PCR amplification of different crops such as GM canola,GM soybean,GM maize,GM rice,and GM cotton.The results showed that the established multiplex qualitative PCR of GM canolas showed higher specificity,and the LOD was 0.05%.(3)Even specific multiplex quantitative PCR detection technique for GM canolas:Specific primers and probes were designed for the flanking sequences of GM canola RF1,Topas19/2,Oxy235 and RF3 lines.Multiplex real-time fluorescence quantitative PCR methods for GM canolas were developed.The LOD of multiplex real-time fluorescent quantitative PCR was determined by using the standard curve method.The specificity of primers and probes were tested by PCR amplification using different representative crops such as GM canola,GM soybean,GM maize,GM rice,GM cotton.The results showed that event specific multiplex quantitative PCR methods of GM canolas were higher specific and sensitive,and LOD was 0.05%.