A Novel Multiplex-PCR Assay To Identify Genetically Modified Soybean Events

Objective: To develop a novel multiplex PCR method mediated by universal primers(UP-MPCR)to quickly and accurately identify twelve genetically modified(GM)soybeans approved for import for food and feed use as raw material in China.Methods: Primers in this study include universal primers,specific primers and chimeric primers.Universal primers were firstly custom designed with the criterias of no homologous sequences to genomes of genetically modified(GM)soybeans and non-GM soybean.Then designed universal primers needed were tested by PCR experiment through the use of genomic DNA of genetically modified(GM)soybeans and non-GM soybean.Only these primers that had no amplified products with soybean templates were used for further applications.Subsequently,three screened universal primers were added to the 5' ends of reverse primer and forward primer of soybean lectin gene and then a pair of long primers was constructed.Amplified products of the long primers were gel purified and then subcloned into the pMD19-T vector.The constructed plasmids were extracted and confirmed by direct sequencing.The sensitivity of the universal primers was evaluated by serial dilutions of this constructed plasmid.Designing gene-specific primers respectively from 5' or 3' flanking sequences of twelve GM soybean events(MON87701,GTS40-3-2,MON89788,CV127,A2704,A5547,DP356043,DP305423,MON87769,MON87708,305423×40-3-2,MON87701×MON89788 and two of them are gene stacked)according to GMDD and patents.Then chimeric primers were obtained by adding the optimal universal primer to5' end of all gene-specific primers.Genomic DNA of GM soybeans and non-GM soybean were used to assess the specificity and sensitivity of those chimericprimers.To establish a multiplex PCR mediated by universal primers for tracking twelve GM soybean events(two of them are gene stacked),ten pairs of chimeric primers specific for twelve GM soybean events were designed.By optimizing the PCR system,UP-MPCR with high Tm in previous cycles and low Tm in followed cycles were set up in this study.Based on the findings of the UP-MPCR in this study,ten DNA fragments separately from twelve GM soybean events and one DNA fragment from lectin gene were subcloned into the pMD19-T vector in different ways to obtain recombinant plasmids used as positive controls.Results: This study have designed and successfully screened three universal primers that have no amplification products from soybeans.The sensitivities of these universal primers are about 100 copies in their detection abilities.In addition,this study also developed 2 plasmids containing the GM soybeans as testing templates.pSOYG1 has 4 fragments that are responsible for MON87701 ?GTS40-3-2?MON89788 and internal control of Lectin.pSOYG2 consists of 5 fragments from A2704-12?DP356043?DP305423?CV-127,and A5547-127.We also developed 2template plasmids that only harbor one GM soybean genes of MON87708 and MON87769.The UP-MPCR system in this study can specifically detect these genomic DNA of twelve GM soybeans.The sensitivities of this system were up to 0.1 ng(the equal of 0.1%(w/w),0.1 ng/100 ng sample DNA).Conclusion: This newly developed multiplex UP-MPCR system can be used to track twelve GM soybean events(two of them are gene stacked)specifically and sensitively.It is novel,accurate,time-saving and low-cost in the detection of GM soybean events.Assays with this new strategy will have a potential in detecting other GM crops.