The Application Of Recombinase Ploymerase Amplification Technology In Transgenic Detection

As more and more GM crops and their products enter the market.This will inevitably put higher demands on their safety regulatory system.In recent years,the rapid development of constant-temperature nucleic acid amplification technology has promoted the application of RPA amplification technology in transgenic detection.RPA technology has good performance,simple operation,convenient equipment and low cost,which attracts high attention and research boom.This experiment aims to establish a rapid detection method for transgenic crops and transgenic products through real-time fluorescent RPA amplification technology,so as to supplement and strengthen China's genetically modified safety supervision system,and provide reference for the establishment of other detection methods.This study mainly consists of the following three parts:In the first experiment,The four most widely used transgenic universal elements CaMV35 s,BT,NOS,PAT were selected by data analysis.Primers and probes were designed based on the specific sequence of the universal components of the transgene,and a 6-tube continuous real-time fluorescent RPA rapid screening system was combined with the freeze-dried powder technique.The system has high specificity and sensitivity,and can effectively detect templates with a minimum of 48 copies.In the screening of actual samples,the screening results are stable and reliable,and have wide application value in the preliminary rapid screening of genetically modified crops and products.In the second experiment,primers and probes were designed according to the transformant specific sequence of transgenic soybean line MON89788,and the RPA real-time fluorescence detection method of transgenic soybean MON89788 was established.Systematic research and analysis were carried out on key factors affecting RPA amplification,such as screening of optimal primers,optimization of primer and probe final concentration,and reaction temperature.The detection method is specific and has an absolute detection limit of 40 copies for MON89788,and the relative detection limit is 0.05%.In the third experiment,primers and probes were designed according to the specific sequence of the transgenic maize line MON863;a real-time fluorescence detection method for transgenic soybean MON863 was established.The reaction temperature and the final concentration of the probe and primer were tested to select the optimal reaction conditions.Condition.The fluorescence of the RPA was monitored in real time using a portable instrument.Omit the purification step by real-time RPA combined with Noah-based DNA extraction method to directly amplify free DNA extract from crude cell lysine,and display the amplification results in real time,provide sample in/out field detection platform,and detect with RT-PCR the results were compared with the time taken for the test.The results of the two methods were consistent.The average detection time of RT-RPA was 8.46 minutes,and the average detection time of RT-PCR was 37.23.RT-RPA takes much less time than RT-PCR.Including 1 minute of preparation of the sample,the entire detection process using RT-RAP is approximately 10 minutes.In summary,the rapid detection method based on real-time fluorescent recombinase polymerase amplification technology shows high accuracy and specificity,short time-consuming and simple operation.This method is not only suitable for on-site screening of genetically modified components,in other many fields also have great application potential.