| The spring up of genetically modified (GM) products have caused widely awareness of foodsafety by public. By now, there is no authorized GM staple crops, but the GM rice occasionally spreadout widely in farmlands. In order to protect consumers’ right to know and options about their products,it is urgent to develop an easy and rapid method to detect GM crops, which is used to strengthen themanagement of transgenic product security. In recent years, isothermal amplification technology isdeveloping rapidly. It can get rid of traditional PCR thermocycle instrument, and obtain a correct testresults in a short of time. Recombinase polymerase amplification (RPA) is a novel isothermal DNAamplification and detection technology, which can complete the amplification in a short period oftime under the condition of constant temperature of39℃by simulating in vivo DNA recombinationreaction within20minutes. Combined with DNA rapid extraction technology, this method has a widerange of applications in wild rapid detection of GM crops in the future. This study has developed aRPArapid DNA detection method and a quick DNA extract technology. The main content and resultare as follows:1. RPA isothermal amplification technology is used for GM detection research: We has selectedseveral widely used target in GM detection, such as CaMV35S promoter, nos terminator, HPT geneand Bt gene et al, and developed RPA rapid detection methods based on agarose gel electrophoresisand fluorescent probe. The result has proved that real time RPA method has the advantage of highsensitivity, and can stably detect as few as100copies of the target molecules in samples. What’smore, real time RPA method has the property of good applicability and is able to detect differentconstituents of GM crops, and can obtain a steady, reliable result in15to25minutes.2. The development of rapid DNA extract method: Through the optimization of extractionconditions, we used three kinds of portable device to developed a rapid DNA crude extract methodcoupled with magnetic bead. This method can extract different plant DNA within20minutes in roomtemperature. The result suggest the crude sample DNA can be used for PCR amplification, and evencan be used to event-specific detection of GM maize MON810at the level of0.1%. Next, combinedwith RPA fluorescence detection technology, we successfully identified the nos terminator and Btgene constituent in GM rice TT51-1, and developed the rapid and accurate wild detection method forGM crops. |
