Study On The Detection Of Herbicide-resistant Glyphosate Soybean And Its Products By RF-SRCA

Since genetic engineering technology has been widely used in crops,genetically modified technology has been developed rapidly.Traits such as herbicide-resistance,insect-resistance,disease-resistance,drought-resistance,and cold-resistance have been introduced into genetically modified crops.Therefore,crop yield has been increased and production cost has been reduced.Subsequently,genetically modified crops have made considerable development and been widely used in the world.Nonetheless,the potential impact of genetically modified crops on humans and the environment is still unclear,which has attracted public attention for many years,and the application of genetically modified crops has also caused more and more controversies.In order to protect consumers' right to choose and right to know,mandatory genetically modified labeling regulations have been adopted in many countries and regions to strengthen the management of genetically modified crops.Genetically modified soybeans occupy a dominant position in genetically modified crops,and the most widely used genetically modified soybean varieties are herbicide-resistant glyphosate soybeans.China imports a large amount of genetically modified soybeans from overseas every year,and is the country with the largest amount of import and crush of genetically modified soybeans.Consequently,in order to protect consumers' right to choose and right to know,it is urgent to develop a simple,rapid,sensitive and accurate detection method for genetically modified crops.In this study,a novel real-time fluorescent saltatory rolling circle amplification(RF-SRCA)technology was established for the first time,which combined fluorescence technology with saltatory rolling circle amplification(SRCA)technology,for the detection of glyphosate-resistant soybeans and its products.This method only requires a pair of primers and avoids the artificial cyclization process to achieve high-efficiency amplification of linear DNA under the effect of Bst DNA polymerase,and the amplification results can be obtained by the real-time fluorescence amplification curve.Furthermore,the amplification results can also be analyzed by visualization and gel electrophoresis methods.In this study,the herbicide-resistant glyphosate gene CP4-EPSPS(GenBank No.143998)was selected as the target gene.Then,the specific primers were designed by using Primer Premier 5.0 and DNAMAN software,respectively.A pair of primers with excellent specificity was selected through a large number of preliminary experiments.Meanwhile,the reaction conditions and reaction system of RF-SRCA were optimized,respectively.Finally,the RF-SRCA reaction system was established.The optimal reaction conditions were as follows:reaction temperature(62?),2.5 mM dNTPs(4.0 ?L),20 mM MgSO4(2.5 ?L),10 ×ThermoPol Reaction Buffer(2.0 ?L),10 ?M forward and reverse primers(1.5 ?L each),template DNA(1.5?L),8 000 U Bst DNA polymerase(8 U),fluorescent dye EvaGreen(1.0 ?L),with sterile deionized water added to make a final volume of 20.0?L.The specificity of the primers was verified by using 20 genetically modified crops and 2 non-transgenic crops.As expected,5 transgenic soybeans containing CP4-EPSPS gene presented positive results,15 transgenic crops without CP4-EPSPS gene and 2 non-transgenic crops revealed negative results,indicating good specificity of the primers designed in this study.The genomic DNA of genetically modified soybeans(GTS40-3-2)and artificially spiked samples were extracted by the kit method,and then the RF-SRCA reaction was performed,respectively.The sensitivity and detection limit of the method were measured and compared with real-time fluorescent PCR(RT-PCR)and real-time fluorescent LAMP(RT-LAMP)methods.The results showed that the sensitivity of RF-SRCA method for the detection of genomic DNA of genetically modified soybean were 8.8 × 100 fg/?L through real-time fluorescent amplification curve method and visualization method,and was 8.8 ×101 fg/?L by RF-SRCA gel electrophoresis method.The sensitivities were 8.8 × 102 fg/?L and 8.8 × 101 fg/?L by RT-PCR and RT-LAMP,respectively.In artificially spiked samples,the detection limits of RF-SRCA were 0.01%by real-time fluorescent amplification curve method and visualization method,and was 0.05%by RF-SRCA gel electrophoresis method The detection limits of RT-PCR and RT-LAMP were 0.1%and 0.05%,respectively.Hence,the sensitivity of RF-SRCA by real-time fluorescent amplification curve method and visualization method were 10 times higher than RT-LAMP method and RF-SRCA gel electrophoresis method,and were 100 times higher than RT-PCR method.The sensitivity of RF-SRCA by gel electrophoresis method was 10 times higher than that of RT-PCR,which was the same as RT-LAMP method.The detection limit of RF-SRCA by real-time fluorescent amplification curve method and visualization method were 10 times lower than that of RT-PCR method and 5 times lower than that of RT-LAMP method and RF-SRCA gel electrophoresis method.RF-SRCA by gel electrophoresis method had the same detection limit as RT-LAMP method.Moreover,68 commercially available soybean products were tested using RF-SRCA and industry standard method(SN/T 1204-2016)in this study.According to the industry standard method,the results showed that the sensitivity,specificity and coincidence rate of RF-SRCA method were 100.00%,98.41%and 98.52%,respectively.In summary,the RF-SRCA method established in this study can quickly,sensitively,accurately and efficiently detect genetically modified components in soybean products,which provides a good technical platform for the detection of genetically modified crops.