Establishment Of A Field Visual Detection Method For Genetically Modified Crops By Fluorescence RPA

Among the nucleic acid-based detection methods,PCR(such as common PCR,real-time PCR,etc.)is the largest number of transgenic detection methods developed at home and abroad,and has been used as the standard inspection method of relevant food laws and regulations in many countries.However,the PCR method relies on precision instruments and is time-consuming,which limits its use in field detection.In this study,based on recombinant polymerase isothermal amplification(RPA)technology,transgenic corn and transgenic soybean were taken as the research objects to establish a nucleic acid detection technology that can be used in the field.It is simple and quick,with strong specificity,short time consuming and low cost.From the extraction of DNA to the detection of nucleic acid amplification results,it can be completed in the field.The main research contents and results are as follows.1.According to the specificity of the silicon membrane to adsorb DNA,combined with the filter membrane and the syringe,a method for rapid extraction of plant genomic DNA can be established on-site.This method does not need to use liquid nitrogen or a centrifuge,and the DNA extraction is completed within 5 minutes.The extracted DNA from the seeds and leaves of five crops such as soybean,cotton,rapeseed,corn and rice were subjected to ordinary PCR and ordinary RPA amplification of 18 Sr RNA endogenous genes,with good results.2.Using ordinary PCR,fluorescent RPA and fluorescent quantitative PCR to simulate the actual detection of genetically modified soybean SHZD32-1 DNA extracted by rapid DNA extraction method,the detection results of the three methods are consistent,and the genetically modified components of soybean SHZD32-1 can be accurately detected.3.Based on fluorescent RPA,with transgenic corn double antibody 12-5 as the research object,a visual detection method was established that irradiated the amplified product with a handheld blue light and directly analyzed and judged the amplified product with the naked eye wearing protective goggles.The established transgenic corn double antibody 12-5 visual detection system has strong specificity,and the detection sensitivity can reach 10 copies.4.Fluorescent RPA has good temperature adaptability.The method of using warm stickers and heating packs to heat the sample tried in this experiment can complete the amplification of the sample in 20 minutes,and the amplified product can be used in the field with the visual detection method.5.In this study,the rapid DNA extraction method was combined with the fluorescent RPA visual field detection method,and the genetically modified soybean SHZD32-1 was used as the research object to establish a complete field rapid detection technology from DNA extraction to nucleic acid amplification result detection,and actual detection verification was carried out.The results show that the established visual detection system for genetically modified soybean SHZD32-1 has strong specificity,and the detection sensitivity reaches 10 copies.The actual detection result is consistent with the detection result of q PCR method.It not only meets the needs of rapid on-site detection of genetically modified soybean SHZD32-1,but also The development of other on-site rapid detection methods provides new ideas.