Specific Qualitative PCR Detection Method for Transgenic Maize Zheda Ruifeng 8
[Objective]Zheda Ruifeng 8 is an insect-resistant transgenic maize developed by Hangzhou Ruifeng Biotechnology Co.Ltd.,the safety certificate for production and application was obtained in December 2021.The specific PCR detection method for Zheda Ruifeng 8 was investigated to provide technical support for market regulation and industrialization.[Method]In this study,the flanking sequence of the insertion end of exogenous gene of Zheda Ruifeng 8 event was used as the target sequence,specific primers were designed at the 5'-end(right flanking)and the 3'-end(left flanking)respectively.Twenty pairs of primers for each of the left flanking and the right flanking were screened by the standard reaction system and the reaction procedure that was consistent with that of the standard gene zSSIIb in maize,and the primers with high specificity and fine stability were obtained.A preliminary method for the detection of PCR of the Zheda Ruifeng 8 event was established by changing the two key parameters of the annealing temperature and the concentration of the primer.[Result]The screened Zheda Ruifeng 8 PCR amplification primer RF 8-RB-R4/F1 may specifically amplify the target fragment of 265 bp,and a standard reaction system and reaction procedure consistent with the standard gene zSSIIb in maize was established,and it achieved high throughput for batch detection.[Conclusion]The established specific qualitative PCR detection method demonstrated excellent stability and adaptability.It could consistently detect a copy number fraction as low as 0.1％,equivalent to approximately 20 copies.This method proved to be highly accurate and specific,effectively detecting the transformants of Zheda Ruifeng 8.
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