Onsite rapid detection method for genetically modified maize and soybean based on recombinase polymerase amplification.

Abstract

With the commercial planting of transgenic maize and transgenic soybeans in China, in order to ensure the healthy development of China's biological breeding industry, it is urgent to develop on-site rapid detection techniques for transgenic maize and soybeans. In this study, a rapid detection method based on recombinase polymerase amplification (RPA) was proposed to overcome the shortcomings of on-site rapid detection of transgenic maize and soybean. Representative transgenic maize (Shuang Kang 12-5, DBN9936, MON810) and soybean (DBN9004, GTS40-3-2, SHZD32-1) were selected as test objects. According to their flanking sequence, specific primers and probes were designed and screened, and a RPA amplification system with high specificity and sensitivity of 20 copies for each transgenic event was established. In order to quickly obtain DNA, seed samples use nucleic acid release agents to obtain templates that can be used for RPA amplification. In order to eliminate the interference of chlorophyll in leaves on RPA fluorescence signal and quickly obtain DNA, leaf samples use the nucleic acid rapid integrated extraction device developed by our laboratory to quickly obtain DNA and eliminate the chlorophyll interference. Combined with a portable metal bath for initiating RPA amplification, a blue light gel cutter for visual detection, and a rapid on-site detection method for transgenic maize and soybean was constructed. The entire detection process required approximately 20 minutes, and the detection result was consistent with the result of conventional PCR. It is suitable for on-site rapid detection without large equipment, and has strong practicability and adaptability. The proposed approach provides technical support for on-site detection of transgenic crops and offers a reference for the application of other rapid nucleic acid detection technologies.