Multiplex PCR Detection Of GM Cottonseed And Edible Grease

With the rapid development of transgenic technology,there were more and more genetically modified organisms(GMO) and their products in the world.Genitically modified food contain new genetic materials inclouding exogenous DNA and protein.However,the safety issue arose internationally. Many countries adopt the labeled system towards the genetic modified food.The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified food.Multi-plex PCR is successfully established by targeted genes which include CaMV35S promoter,Nos terminato,NPT Ⅱ,CP4-EPSPS and endogenous gene Lectin.DNA was isolated by the improved method and then amplifed by PCR. Five genes are simultaneously specifically detected.Commercial conttonseed,genetically modified conttonseed and oil were detected with the five-plex PCR.The linear range of the method of detection is0.1%.In present study,The reaction mixture consisted of1μL of Taq(5U/μL of stock solution),5μL of10×PCR amplification buffer,4μL of dNTPs(1mM each in solution),0.5μL of each primer(10pmol stock solution) of forward,0.5μL of each primer(10pmol stock solution) of reforward,each DNA templates1μL and20μL of double-distilled water.The reaction was run under the following conditions:Cool start;DNA pre-denaturation at94℃for5min;DNA denaturation at94℃for60s,primer annealing at56.7℃for30s,and DNA extension at72℃for60s for35cycles;the last extension was performed at72℃for5min.The PCR products were examined by electrophoresis with2%agarose gel.The amplified product was sequenced and compared with CaMV35S、Nosl、GUS、CrylAc and tRNALeu in Genebank and found to have the homogenicity of100%、00%、100%、100%、100%.Furthermore,Multi-plex PCR is a feasible and effective genetically modified conttonseed and contton product detection method and will have broad application prospect.