Comparative Study On The Detection Of Rice TGW6 Gene Editing

Gene-editing technology has become one of the most popular technologies in the field of biology.The screening and identification of individual gene editors has become an important research content.In this study,a new detection method based on qPCR platform was developed.The sensitivity and specificity of the qPCR detection method were tested with 9 plasmids as samples.The reported methods of T7 endonuclease I?T7E1?,annealing at critical temperature PCR?ACT-PCR?and high resolution melting curve?HRM?were compared comprehensively.The limitations of other methods in time,sensitivity,labor force,cost and sequence were found,which highlighted the advantages of qPCR in these aspects.Further,the rice edited by Nanjing46 and TGW6 gene was used as experimental material to detect and identify gene edited rice samples by qPCR and digital PCR.The main results are as follows:1.Detection of sensitivity and specificity by qPCR,Double-probe experiments show that the method can detect DNA samples with single base deletion,insertion and substitution accurately.For the screening of the edited samples of rice TGW6 gene,qPCR method can accurately identify and distinguish the types of samples.The results of calculation and analysis of a large number of sample data by 2^-??CTmethod show that the mutation rates of wild type,heterozygote and homozygote are about 1?1.16 to 0.80?,0.5?range from 0.64 to 0.41?and 0.2.DNA samples of 9 synthetic plasmids were detected and identified by T7E1 restriction enzyme digestion,ACT-PCR and HRM.After 5 hours of restriction enzyme digestion by T7E1restriction enzyme digestion,the results were good.Two bands of single base mutants were cut out and the bands were obvious.In the ACT-PCR detection method,the annealing temperature was set to 73 degrees.Gel electrophoresis showed that only WT wild type bands appeared,but no mutants were found in the mutants.HRM analysis method is also suitable for screening mutants of gene editing,but this screening method can not distinguish single base substitution of A-T type.3.T7E1 digestion method and ACT-PCR method consume more than 4 hours in time,while HRM method consumes less than 2 hours in time.Compared with the other two methods,the sensitivity of T7E1 digestion method was medium.ACT-PCR method and T7E1 digestion method have high manpower cost because of their complicated operation,while HRM method is of medium level.Compared with T7E1 digestion method and ACT-PCR method,HRM analysis method has higher cost.ACT-PCR has some sequence limitations,while the other two methods have no sequence limitations.4.Detection of rice editing TGW6 gene by digital PCR,Further extending the qPCR method to ddPCR platform,expanding the sensitivity of the method,and optimizing the experimental links.The method can successfully distinguish wild type,heterozygous type and homozygous type mutations.The results show that the target gene/internal reference gene ratio is 0.999?1?0.512?0.5 and 0.0039?0,implementing the purpose of method upgrade.