Study On PL3 Gene Editing Rice Editing Site Detection Method

With the development of gene editing technology,gene editing crops are emerging.In this study,PL3 gene editing of rice was tested in order to provide technical support for quantitative detection methods of gene editing crops and meet the needs of quantitative identification of gene editing crop products in industrialization.Methods: the PL3 gene editing of rice was used as the research object,and pyrosequencing,HRM(high-resolution dissolution curve)and dd PCR were used as analysis tools to carry out precise quantitative detection of PL3 gene editing of rice.(1)The detection method of pyrosequencing for PL3 gene editing rice target site was established through experiments on the design of pyrosequencing primers for PL3 gene editing rice target site,detection of primer effectiveness,genotyping and stereotyping,genotyping and quantitative detection,and blind sample detection.(2)Through the PL3 rice genotype edit target site HRM effectiveness,primers,primer design analysis,the reaction system,reaction conditions optimization and classification of quantitative test,determine the HRM reaction primer concentrations and annealing temperature is the best combination of 0.4 ?mol /L and 58 ° C,the detection limit of no less than 10%,established the PL3 gene editing rice target site HRM detection method.(3)Through the PL3 genotype editor rice target site dd PCR primers and the probe design,the establishment of the reaction system,the quantitative range test,LOQ validation test,determine the digital primer PCR reaction concentration of 0.4?mol/L,probe concentration of 0.15?mol/L,the best annealing temperature is 62.4?,the minimum detection limit of 0.05%.A dd PCR method for PL3 gene editing in rice was established.The three detection methods established in this study have high sensitivity and accuracy,and can be used for the qualitative detection and accurate quantitative analysis of PL3 gene editing rice target sites.