Development Of Plasmid Reference Materials For Detection Of Genetically Modified Rice Events And Evaluation Of Certified Value Measurement Methods

Reference materials are very significant in GMO product quantitative analysis,however,the development of GM reference materials our country is still in the initial stage.Therefore,development of novel reference materials can solve the deficiency of reference materials.Herein,we constructed six single target plasmid reference molecules which could be applied to detect corresponding genetically modified rice events.And four of them were performed applicability analysis,assessment of homogeneity and stability in real-time PCR.And we also measured the certified value of plasmid reference materials by digital PCR platforms.1.In this study,we developed six single target plasmid reference molecules successfully,which all contained the tandem integration of a rice endogenous SPS fragment(291 bp),a 2463 bp DNA fragment and a corresponding event-specific fragment.The results from applicability analysis indicated the standard curves using these four plasmid reference materials as calibrator had high PCR efficiency(95%-102%)and good linearity(0.9994-1.0000).Furthermore,all standard curves had good repeatability and reproducibility,the relative standard deviation of repeatability ranged from 0.01% to 1.26%,and the relative standard deviation of reproducibility ranged from 0.13 % to 2.84%.And the four reference plasmids had good sensitivity in the qualitative PCR analysis,which LODs were 5 to 10 copies per microliter and LOQ were low to 10 and 20 copies per microliter.With the correction of Cf value,the quantification bias of simulated samples varied from 0.25% to 24.1%,and the relative standard deviations were from 3.11% to 25.96%.The above mentioned values were below the accepted level of the related guideline.From the result of homogeneity tests,all plasmid reference materials were good in homogeneity.And from the data of assessment of stability,the plasmid reference materials could be stored at least half a year stably.Therefore,the developed plasmid reference materials are acceptable and suitable for quantification of the GM rice events.2.With the increasing development of genetically modified organism(GMO)detection techniques,digital PCR which are independent of carlibrant,shows huge application prospectin GMO product detection,espiecially in the certified value measurement.We mesured the certified value of plasmid reference materials by three digital PCR platforms(cd PCR,dd PCR and 3D dPCR).The results from three digital PCR platforms are consistent and close to the theoretical value,1 copies/copies.Therefore,digital PCR technology is a novel method suitable for measuring the certified value of reference meteriels with high accuracy and may be widely employed in the future.