Research And Application Of Digital PCR In Accurate Quantification Of GMOs

Digital PCR is an absolute quantitative detection method that does not depend on standard curves and reference materials.Because of its high sensitivity and good accuracy,it is widely used in the quantitative detection of genetically modified components and the development of reference materials for genetically modified detection.Although digital PCR can accurately quantify nucleic acids,there are still significant differences in measurement results between different operators and different laboratories in the actual measurement process.By using a nucleic acid molecule with an accurate copy number concentration or copy number ratio as a quality control tool Aids in detection accuracy.In this study,the laboratory developed screening elements containing 11 commonly used transgene detection(P-Ca MV35S,P-FMV35S and P-NOS three promoters,Bar,NPTII,HPT and Pmi four marker genes,T-NOS,T-35S,T-g7 and T-e9 four terminators)and standard gene sequences in maize,soybean,rapeseed,rice,cotton and wheat,transgenic rice SD-rice,transgenic rape SD-rape and p BI121-Screening plasmids are Research materials,using digital PCR to conduct accurate quantitative research on genetically modified components,the main research contents are:1.Identification of SD-rice genotype of transgenic rice.According to the whole genome sequencing information of transgenic rice SD-rice and the gene sequence information of the deletion of the receptor insertion site,2 pairs of PCR detection primers were designed,and one pair of primers was designed on the gene sequence near the insertion site(SD-R1023),a primer was designed on the gene sequence with the deletion of the insertion site(SD-QF715),and the amplification length was 308 bp.For the other pair,one primer was designed on the gene sequence near the insertion site(SD-rice F255),and the other was designed on the inserted gene sequence(SD-rice R366),and the amplification length was 112 bp.A single-copy homozygous transgenic rice SD-rice material was identified by qualitative PCR amplification and agarose gel electrophoresis.2.Establishment of event-specific detection methods for transgenic rice SD-rice and transgenic rape SD-rape.Based on SD-rice F255/R366,the Taq-man probe SD-rice P303was designed,and a event-specific real-time quantitative PCR detection method for SD-rice transgenic rice was established.The standard curve was:Y=-3.411X+37.979(R~2=0.999).The SD-rape119F/272R/242P primer-probe combination was screened out,and event-specific real-time quantitative PCR detection method of transgenic rape was established.The standard curve was:Y=-3.215X+38.840(R~2=0.995).The specificity and sensitivity of SD-rice and SD-rape event-specific fluorescence quantitative PCR detection methods were analyzed,and the results showed that the established event-specific detection methods had good specificity,with an LOD as low as 5 copies/μL and a LOQ of 40copies/μL,which can be used for accurate quantification of transgenic rice SD-rice and transgenic rape SD-rape.The specific digital PCR detection methods for SD-rice and SD-rape were optimized in terms of annealing temperature,primer-probe concentration and so on.The copy number ratio of exogenous gene and endogenous reference gene in SD-rice was 0.924,and the copy number ratio of exogenous gene and endogenous reference gene in SD-rape was 0.474,both close to the theoretical value.3.Verification of the accuracy of digital PCR measurement results.Plasmid DNA and genomic DNA contains 11 commonly used transgene detection screening elements(P-Ca MV35S,P-FMV35S and P-NOS three promoters,Bar,NPTII,HPT and Pmi four marker genes,T-NOS,T-35S,T-g7 and T-e9 four terminators),and SD-rice,SD-rape,plasmid p BI-Screening of the internal reference genes of maize,soybean,rapeseed,rice,cotton and wheat were used as experimental materials.The copy number concentration and copy number ratio of the gene were studied using the same DNA(genomic DNA or plasmid DNA)as a template,using different detection methods(different detection targets or different primer-probe combinations of the same detection target).Meanwhile the same detection methods were used in different types of DNA for digital PCR amplification.The obtained copy number concentration and copy number ratio were counted to assess the effect of template type and detection method on accuracy.The results show that for the same detection target,there are significant differences between the quantitative detection results of different detection primer-probe combinations,so the method needs to be fully evaluated when selecting a quantitative detection method.By calculating the difference between the copy number ratio of the detection target and the internal standard gene and the theoretical value,the optimal detection method of each detection target was screened out.SD-rice and SD-rape genomic DNA,non-transgenic rice and non-transgenic rape genomic DNA,and p BI-Screening plasmid DNA were diluted to 3.7×10~4copies/μL,respectively.SD-rice DNA was mixed with non-transgenic rice DNA to prepare SD-rice gradient blind samples S1-G-rice～S4-G-rice;SD-rape DNA was mixed with non-transgenic rape DNA to prepare SD-rape gradient Blind samples S1-G-rape～S4-G-rape;the plasmid solutions were mixed with non-transgenic rice to prepare blind samples S1-P-rice～S4-P-rice;the plasmid solutions were separately mixed with non-transgenic rice Rape DNA was mixed to prepare blind samples S1-P-rape～S4-P-rape.The gradient samples of the above 4 concentrations correspond to the transgene content of5%,1%,0.5%and 0.1%,respectively.The above blind samples were quantitatively detected by real-time fluorescence quantitative PCR and digital PCR.P-Ca MV35S and T-NOS were used as exogenous gene detection targets,and PLD and Cru A were used as endogenous reference gene detection targets,and the copy number ratios were calculated.The results showed that the accuracy and precision of the quantitative results of the above four types and four gradient blind samples on the digital PCR platform met the requirements(Bias and RSD were both less than 25%).On the real-time PCR platform,genomic DNA reference materials can accurately quantify genomic DNA samples,while plasmid DNA reference materials cannot accurately quantify genomic DNA samples.Neither genomic DNA nor plasmid DNA standard materials can accurately quantify DNA blind samples formed by mixing plasmid DNA and genomic DNA.It is proved that there is a matrix difference between genomic DNA molecules and plasmid DNA molecules,which affects the detection results of real-time quantitative PCR,but has no effect on the detection results of digital PCR.4.Determination of droplet volume of QX200 digital PCR instrument.The 1088droplets generated by the Bio-rad droplet generator were imaged,observed and photographed using an Olympus BX53 optical microscope,and the images were processed and analyzed by Image J software.The volume of droplets produced was calculated by three methods,and the measurement uncertainty was evaluated.The average droplet volumes calculated by the three methods were 0.772±0.02 n L,0.75±0.04 n L,and 0.796±0.05 n L,respectively,the total average droplet volume was 0.772 n L,and the measurement uncertainty was 1.15%.Determining the volume of digital PCR droplets in each independent laboratory can help reduce errors in digital PCR measurement results and ensure the accuracy of digital PCR quantitative results.In this study,a method for identifying genotypes of genetically modified rice SD-rice was established,and a specific detection method for genetically modified rice SD-rice and genetically modified rape SD-rape transformants was established and optimized,and 11commonly used transgenic detection elements were screened.The optimal detection primers were screened.The probe combination was used to study the effect of DNA type on the quantitative detection results,and the droplet volume was determined.This study provides a strong theoretical basis and scientific basis for obtaining accurate quantitative detection results of transgenes.