Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste,direct quantitative PCR(qPCR)kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study.A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories.Compared with 2 commercial direct qPCR kits,conditions of DNA releasing procedure and PCR amplification were optimized.Element screening was performed at the initial step of genetically modified(GM)ingredient testing procedure via direct qPCR.GM event identification was carried out in positive samples by initial screening.Totally 5 screening elements(P–35S,T-NOS,Cp4-epsps,bar and pat)for soybean materials and 6 screening elements(P–35S,T-NOS,NPTII,Cry1Ac,bar and pat)for cotton samples were detected.In GM event identification,MON531 and MON1445 were found in cotton materials.Results were further confirmed by real-time PCR with DNA extraction and purification.The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.

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

[Objective] This study aimed to establish a multiplex PCR system for de- tecting 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 could 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 2.5μl of 10xPCR buffer, 2.0μl of MgCI2 （25 mmol/L）, 2.0 μl of dNTP mixture （2.5 mmol/L of each dNTP）, 1.0 μl of actin gene primers （10μmol/L）, 1.0μl 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 （10μmol/L）, 0.1 μg of template DNA, 1.25 U of Taq DNA polymerase; ddH20 was added to the total reaction system of 25μl. The PCR reaction program consisted of pre-denaturing at 94℃ 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.