International Validation Of Rice Endogenous Reference Gene Detection Systems And Development Of PCR Detection Methods Of Four Varieties Genetically Modified Cottons

To ensure the implement of genetically modified organisms (GMO) labeling regulation in the world and perfect the detection methods system for GMOs, two aspects that related to GMOs identification and quantification are covered in this study.1. The international validation of SPS gene as rice endogenous reference gene for qualitative and quantitative PCR analysis.Endogenous reference gene is very important for GMOs detection and quantification. One rice (Oryza sativa) gene, Sucrose Phosphate Synthase (SPS), had been proved to be suitable for the endogenous reference of genetically modified (GM) rice detection and quantification in our previous study. Herein we invited twelve GMO detection laboratories to participate in the collaborative ring trial for international validation of the SPS gene as rice endogenous reference gene. In the collaborative ring trial of SPS gene validation, the submitted results from twelve laboratories confirmed the species specificity, less allelic variation and stable single copy number of the SPS gene. Furthermore, limit of detection (LOD) of SPS qualitative PCR was proved to be 0.1%, and limit of quantification (LOQ) for quantitative PCR system was at least 23 copies haploid rice genomic DNA with high PCR efficiency and linearity. Additionally,the bias between the test and true values of eight blind samples ranged from 5.22% to 26.52%. All the validated results indicated that the SPS gene is suitable for using as endogenous reference gene for GM rice and its derivates identification and quantification.2. Qualitative and quantitative PCR methods for event-specific detection of GM cotton.Event-specific PCR is becoming the most effective and important method for GMO detection and quantification. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of Mon15985 and the 3'-integration junction sequence of Mon88913 were revealed. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence and the event-specific qualitative and quantitative detection methods were firstly established for GM cotton Mon15985 and Mon88913. The qualitative PCR method is high specific and sensitivity. The limit of detection (LOD) of qualitative was at least 0.05%. The limit of detection (LOD) and the limit of quantification (LOQ) for event-specific quantitative PCR assay was determined to be approximately 10 and 17 copies haploid cotton genomic DNA, respectively. Furthermore, five researchers were invited to validate the developed event-specific quantitative PCR assays. All results in the in-house validation proved the high PCR efficiency and linearity of the event-specific quantitative PCR system. Additionally, the established quantitative PCR system were examined with five mixed cotton samples with known GM cotton contents. The bias between the true and quantified value were all less than 25%. Therefore, the two developed event-specific qualitative and quantitative PCR methods were applicable for the identification and quantification of GM cotton Mon15985 and Mon88913 and their derivates. And then, based on the previous study about the event-specific detection methods of two other GM cotton samples (Mon1445 and Mon531) and the established event-specific detection methods of GM cotton (Mon15985 and Mon88913) in this study, the multiplex qualitative PCR system was systematically optimized and developed for four GM cotton events. The LOD of multiplex qualitative PCR system was 0.1% with high specificity. Obviously, multiplex PCR analysis has the advantages of improving the efficiency of PCR detection methods and saving the cost of PCR reaction.