Qualitative And Quantitative PCR Detection Of Two Transgenic Rice

The event-specific qualitative assay with conventional qualitative PCR for GM rice Tic-19based on5’junction sequence was established. Through the screening of transgenic elements of this sequence and design of the qualitative PCR primers Tlc-19f/r, Tlc-19strain of the insect-resistant rice specificity of conventional qualitative PCR method. According to the transfer of genes in rice T2A-13’flanking sequence information, get the3’ end of a specific structure.And the qualitative PCR primersT2A-1f/r was designed. Insect-resistant rice Tlc-19and T2A-1specificity of conventional qualitative PCR method was established.By the gos9of the internal standard gene, strain-specific detection, and qualitative PCR sensitivity test results shows that the qualitative PCR detection system is stable, the test results are reliable. Qualitative PCR detection limit was0.01%. Application of the method of overlapping PCR containing Tlc-195’flanking sequence and T2A-13’flanking sequence, respectively, and the internal standard gene in rice gos9sequence two specific fragments of connections was transformed into the pMD-20T vector used to construct standard plasmid named pMD-lc and pMD-2A.Using standard plasmid molecules as a standard substance, rice of genetically modified Tlc-19and T2A-1real-time quantitative PCR method were established.Through the analysis of standard curve regression equation, a good linear relationship between DNA content and cycle threshold showed that the standard plasmid molecules can be used for the quantitative detection.For the known5%,0.25%or5%,0.5%of the two different transfer the gene content of the mixed sample quantitative detection, accuracy of test results deviate from the rate (bias), the accuracy of the standard deviation (SD), relative standard deviation (RSD) were within25%also shows that the reliability of the quantitative detection. Concluded from all above results, the qualitative and quantitative PCR assays were reliable and accurate for Tlc-19and T2A-1measurement.