Establishment of a Multiplex PCR System for Detecting Transgenic Ingredients from Citrus

[Objective] This study aimed to establish a multiplex PCR system for detecting transgenic ingredients from Citrus.[Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citrus,the primers specific to CaMV35S promoter,NOS promoter,NOS terminator and actin gene were designed,to establish a multiple PCR system which can detect four types of sequences.In addition,orthogonal tests were performed to determine the optimal concentrations of all the components in PCR reaction system,as well as the optimal PCR cycle parameters.[Result] The optimal PCR reaction system should contain 2.5 μl of 10× PCR buffer,2.0 μl of MgCl2(25 mmol/L),2.0 μl of dNTP mixture(2.5 mmol/L of each dNTP),1.0 μl of actin gene primers(10 μmol/L),1.0 μl of 35S promoter primers(10 μmol/L),1.5 μl of NOS promoter primers(10 μmol/L) and 0.5 μl of NOS terminator primers(10 μmol/L),0.1 μg of template DNA,1.25 U of Taq DNA polymerase;ddH2O was added to the total reaction system of 25 μl.The PCR reaction program consisted of pre-denaturing at 94 ° C for 5 min;31 cycles of denaturing at 94 ℃ for 30 s,annealing at 64.1 ℃ for 45 s and extension at 72 ℃ for 50 s;final extension at 72 ℃ for 10 min.The reaction system optimized with the orthogonal tests could detect as less as 0.1% transgenic component in the tested samples.[Conclusion] The MPCR detection system established in this study can meet the requirements in theory for detecting the genetically modified ingredients in Citrus or the deep-processed products.