| Due to the rapid development of transgenic technology in my country,transgenic crops are planted in a large area,and the safety of transgenic products has attracted much attention from the society.The implementation of GMO safety management and labeling system require standardized testing methods and GMO testing standard materials.Reference material is an indispensable guarantee for GMO testing.It is of great significance to implement safety management regulations and safeguard national interests in international trade.However,the lack of reference materials has become a bottleneck for the development of reference materials.In this study,a new reference material was developed by cloning exogenous genes and a new reference material development strategy was developed.1.Preparation of reference material candidates.1)Cloning of the exogenous insertion sequence of MON810 maize.Initially determined through the GMDD website,primers were designed on the maize genome on both sides of the insertion sequence,and the full-length fragment of the transgenic maize MON810 insertion was screened by PCR and agarose gel electrophoresis experiments,and the seven amplified fragments that met the expectations were sequenced by ligating the vector to screen the fragments with correct sequences.The target fragment was digested with Ase I and verified by agarose gel electrophoresis.The fragment was then purified and recovered.2)Preparation of negative genomic DNA.Three methods of B73 maize genomic DNA extraction were compared.After quality testing,the concentration and purity of maize DNA extracted by the Tiangen kit method met the experimental requirements.2.Establishment of microdrop digital PCR method.The final reaction strip system was determined and the annealing temperature was optimized,and finally 56°C was screened as the optimal annealing temperature for this experimental study.The microdrop digital PCR reaction procedure was: pre-denaturation at 94°C for 10 min,denaturation at94°C for 30 s,annealing at 56°C for 1 min,45 cycles,extension at 98°C for 10 min.3.Comparison of internal reference genes.The nine internal standard genes of corn hmg,adh I-1,adh I-2,ivr I-1,ivr I-2,z SSIIb-1,z SSIIb-2,10 k Da-1,10 k Da-2 were compared by digital PCR experiments.The results showed that the copy numbers of different internal reference genes were different under the same experimental conditions and the same sample conditions,and no positive droplets appeared in ivr I-2,and z SSIIb-1 negative droplets did not separate from positive droplets,and the rain at 10 k Da-1 was severe.Finally,it was found that the copy number of adh I-1 was stable without non-specific amplification,and the number of raindrops was small,which was used as the internal reference gene in this study.4.Reference material characterization.A gradient dilution of MON810 DNA was performed using B73 maize negative DNA,and the ratio of MON810 copy number to adh I-1 copy number was close to 100% when diluted to 70,000 fold.The standard substances studied in this experiment were calibrated by six laboratories and the results showed that the RSD was between 2% and 3%,which were in accordance with the standard of less than 25% RSD.The uniformity,stability and uncertainty of MON810 mixed reference material were tested and analyzed.The results showed that this standard has good inter-and intra-unit homogeneity and can be transported and stored at room temperature for14 days.In this study,the MON810 mixed reference material was developed to solved the problem of lack of raw materials,and successfully constructed a droplet digital PCR method,which provided a technical reference for the development of reference materials in transgenic and related fields. |
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