Studies On Reference Materials For Detection Of Genetically Modified Organisms

Reference material (RM) for detection of Genetically modified organisms (GMO) is a material orsubstance one or more of whose property values are sufficiently homogeneous and well established tobe used in GMO detection for the calibration of an apparatus, the assessment of a measurement method,or for assigning values to materials. GMO detection RM is indispensable to GMO safety control, GMOqualitative and quantitative detection, and standardization of GMO detection method. It can be used toimprove the comparability, validity and traceability of GMO detection. Procedure of GMO RMpreparation is cumbersome and technically demanding. By now, Only a few departments havedeveloped GMO CRM, and most of the GMO CRM have been developed by EU Joint Research Centre(Institute for Reference Materials and Measurements,IRMM) and American Oil Chemists' Society(AOCS). In China, research and development of GMO detection RM is still in infancy. Lack of RM inroutine work has hindered standardization of GMO detection technology and the implementation ofexisting standards. Therefore, studies on GMO detection RM will promote the development oftechniques and methods for GMO detection, provide technical support for the construction of theregulations of GMO detection, and lay a foundation for developing China's GMO detection RM,towards a international level.In addition, with the development of transgenic technology, varieties and traits of GM plant weremore and more diverse. If we directly detect the blind sample with event-specific method, the testingcost and effort are unacceptable; also it is possible to miss out un-approved GMO. Using screeningdetection as the first step and optimizing the combination of the detection targets with certain geneticelements can greatly improve the detection efficiency. Screening results can also provide a scientificreference for event-specific detection.On the status quo of GMO detection, the dissertation here is composed of two main parts. One is todevelop matrix RM and plasmid RM for detection of GM rapeseed OXY235, Topas, T45, GM soybeanMON89788, GM maize MIR604, T25. And the other is to collect the information of inserted geneelements for the internationally commercialized events of transgenic rapeseed, maize, soybeans and riceto establish molecular characteristics matrix of these transgenic events.The major research contents and the points of innovation in the study are as follows:1. Development and improvement of technology for production of GMO matrix RMThrough the study of GMO detection matrix RM, we have solved the key technical issues in rawmaterial identification, processing, evaluation of homogeneity and stability, determination of value andcalculation of uncertainty. Based on identification of raw materials, the seed were dried under constanttemperature at40℃, and kept water content below10%. The seeds were milled using a cryo-grindingvibrating mill to ensure that more than80%of particles less than200uM. Four transgenicconcentrations were prepared according to the weight and water content of GM and Non-GM material.Bottling was implemented by automatic devices. Random samples of the prepared RM were taken, and GM content was analyzed using real-time PCR. Homogeneity and related uncertainties were calculated,indicating that these RMs were homogeneous. Short-term stability was studied at4°C,25°C and37°Cfor1,2,4weeks, and long-term stability at4°C and-20°C for1,2,4,6months. The results indicatedthat these RMs can be shipped under ambient conditions and stored at4°C or-20°C for6months ormore. At last, the GM content was determined by inter-laboratory study, and the expanded uncertaintyof certified value was comprised with homogeneity, long-term stability and inter-laboratory studystandard uncertainty. A complete set of the techniques for preparation of matrix RMs have beenesdablished.2. Preparation of matrix RM for three transgenic events, each event with4concentrationsThe matrix RMs was prepared for GM events of soybean MON89788, maize MIR604and T25.For each event, four concentrations (0%,0.5%,1%, and5%), and100bottles for each concentrationwere prepared. Through determination of value and uncertainty, these RMs were proved to besufficiently homogenous and stable, and can be used in GMO qualitative and quantitative detection.3. Development and improvement of technology for production of GMO plasmid RMA strategy to construct plasmid molecule, and to test the suitability and commutability of theconstructed plasmid in PCR assays has been established. In plasmid vector construction, firstly, theflanking sequence, endogenous reference gene and interval sequence were determined according toexisting standard detection methods. Secondly, these three fragments were clone into pBluescript II SK(+) vector through molecular biology methods, such as PCR, restriction enzyme digestion and ligation.Finally, the vector was confirmed by PCR, enzyme digestion and sequencing. The certified value ofplasmid RM was the number of cloned DNA fragment for the flanking sequence and endogenousreference gene targets per plasmid. In addition, the specificity and sensitivity of plasmid template inqualitative PCR assays was test, similar results were obtained when using pDNA and gDNA as template.In order to study the commutability of pDNA in quantitative PCR, standard curves of pDNA and gDNAwere constructed and compared; both of these standard curves were used to calculate conversion factorand conversion equation, and to measure GM content of blind samples. We put forward a new andfeasible solution for commutability of pDNA in this study.4. Preparation of plasmid RMs for six transgenic eventsSix different plasmid reference molecules were constructed for GM rapeseed OXY235, Topas, T45,soybean MON89788, maize MIR604and T25. The plasmid DNA was diluted to a concentration107copies/μL, and filled500μL in to a bottle,1000bottles of plasmid RM were prepared for each plasmid.The experiment results proved that the plasmid RM have a good homogenous and stability, and can bewell amplified in PCR assays. So plasmid DNA is good as a RM for GMO detection.5. Construction of the inserted gene elements matrix table of commercialized transgenic events,and establishment of the screening strategy for GMO detectionMatrix tables were constructed on the basis of target gene, promoter, terminator and select markergene by retrieving Canadian GM crop database, European GMO compass and GMO Detection methodDatabase (GMDD), domestic and foreign commercialized events of transgenic rapeseed, maize, soybean and rice were collected. Based on the frequency of application of these gene elements,P-FMV35S, P-CaMV35S and BAR were used in rapeseed screening detection. The combinations ofscreening detection targets were optimized to be P-CaMV35S and T-NOS for maize, P-CaMV35S,Cry1Ac, gat, T-35S and T-E9for soybean, P-CaMV35S and T-NOS for rice. Using these elements, theGMO can be reliably detected once. This study has established a strategy for transgenic screeningdetection and for rapid identification of GM events, providing a scientific basis and an importantreference for the screening detection of GM crops.