Study Of Rapid Detection Technology For Genetically Modified Products

With the rapid development of transgenic technology, the type and quantity of geneticallymodified (gm) crops have increased dramatically.As more and more foreign insert elements aretransfered into crops, direct detection of some insert components or specific transformationevents become time-consuming, laborious and high cost.So many genetically modified productsinspection agencies abroad to build GM products rapid screening technology, and combined withthe matrix method greatly improve the efficiency of the GM products detection, there are nocertain reports about the rapid screening technologies in our country.Due to differentmanagement of GM products exist in different countries,so that the rapid detection method andselected components are also different.This study is based on the information of exogenous gene,control element,marker gene and T-DNA sequence from China’s import and export GMproducts,to establish a rapid detection method for domestic GM products.The main research is asfollows:1.pCaMV35S and tNOS are preferred parameters in screening detection of GMO：In thisstudy,we collected four pairs of primers,of which product length are respectively195bp,165bp,147bp,123bp for pCaMV35s,and180bp,172bp,165bp,118bp for tNOS.The primersmentioned above are used in different international and domestic standards.The specificity,sensitivity and amplification of8primers were tested and evaluated through ordinary PCR andReal-time PCR.Through the test,the products length of which are respectively165bp and147bpfor pCaMV35S and172bp and165bp for tNOS present higher specificity and sensitivity,theyalso present high efficiency in detecting different processed products.those primers present thebest screening results.pCaMV35S195bp and tNOS180bp producted weak and nonspecificamplification.Compared to other primers,the ampilcation of pCaMV35S123bp and tNOS118bpis weak and unstable.Hence the combination of ordinary PCR and Real-time PCR is carried outto evaluate the primers in different international and domestic standards.a reliable technicalmethod is provided to the detection and supervision of GMOs.2.Study of SYBR Green I method to detect16elements simultaneously for the fastdetection of GM products:in the study,information of foreign genes,regulatory elements, markergene and T-DNA sequence of GM products were collected,through emphasizing on the annealingtemperature and length of amplified fragment,one-off amplification of PCR primers weredesigned or collected.SPS,PLD,Lectin,HMG I/Y,Zein,pCaMV35s,FMV35S,T-35s,E93’,the g7,tNOS,Gus,Cry1Ab,cry2Ab,NptⅡ,PAT,cry2Ab,g7,E9,barnase and barstar21groups.Based onreal-time fluorescent PCR (Real-time) method,testing the specificity and sensitivity of primersand analysing the corresponding amplification curves,dissolve curves,Ct value and normalprimer amplification Tm,so that more specific and sensitive primers were acquired for the rapiddetection of genetically modified products.In specific amplification experiments,SPS,PLD,Lectin,HMGI/Y,Zein,pCaMV35s,E9,FMV35S,T-3’,g7,tNOS,Gus,Cry1Ab,cry2Ab,NptⅡ,PATsuccessfully get amplification, dissolve curve were unimodal dissolution curve, blank control didnot produce the corresponding specific amplification,however,amplification of barnase andbarstar primers are very weak, these weak amplied signals may be lower content of theexogenous gene composition.Therefore,all21primers are specific for their targets.In order todetermine the sensitivity of each set of primers,choose1%transgenic materials were analysedafter a linear serial dilution.Final GM content is less than0.01%.Through test, primers except forsensitivity of cry2Ab,PAT is0.1%,the sensitivity of the rest of primers is0.01%.Calculate theminimum copy number of set of primers,in addition to the minimum detectable copy number ofcry2Ab is42copies,that of PAT is85copies,the rest of the primers are able to detect less than10copies.It can be concluded that,each set of primers in normal amplification presents a fixed valueof Tm (error within1℃),that provides a reliable basis for detecting the samples with loweramounts of GM.3.Research on the expression of Bt gene and other unexpected gene in transgenic ricebased on EST high-throughput sequencing technologies:The EST high-throughput sequencingtechnologies were used to inestigate the profile of gene expression in W1201kemingdao,W1202xiushui11,W1207minghui86,W1208TT51,W1209minghui63,W1109kefeng6.The expectedexpressed genes Bt and unexpected expressed genes were generated in transgenic rice comparedwith its receptor.Bt gene is expressed in all three transgenic rice,especially in W1109kefeng6,the amount of expression of Bt was significantly higher than that in W1201kemingdao andW1208TT51.However, there was no expression in the non-transgenic rice,so the detection of Btgene was supported by the profile of gene expression.These differentially expressedgenes,according to their main functions,were divided into Biological process,Cellular componentand Molecular function.According to the main Metabolism pathways,these differentiallyexpressed genes were divided into Metabolism,Genetic Information Processing and CellularProcesses.Eventually the expression differences in different biological functions and ofmetabolic pathways were determined.This can provide significant evidence for predicting thetraits and effects of unintended differential expression genes.