The Application Of RPA Technology In Detection Of Transgenic Maizes

With the innovation of transgenic technology,a wide variety of new genetically modified crops(GMCs)appeared.Comparing to its counterpart,GM maize were improved in both yield and quality.As an important food and feed crop,the planting area of GM maize increased from 0.3 million hectares to60.6 million hectares between 21 years from 1996 to 2016.Social public pay more attention to safety of GM maizes,at the same time,countries have enacted the laws and regulations to monitor GM maize.In order to strengthen the safety management of GM maize and protect consumers' rights to know,rapid detection methods of GM crops are necessary.The major traditional detection methods used polymerase chain reaction(PCR),which still has a lot of disadvantages,such as long reaction time,high cost,special requirements for specific reaction and complicated program.Therefore,researchers begin to focus on isothermal amplification technology.Novel developments provide the possibility of amplifying DNA without the need of a thermocycling machine and get accurate results in a short period of time.Recombinase polymerase amplification(RPA)simulate the progress of DNA replication in living cells,and amply nucleic acid external within 20 minutes under the condition of temperature of 37-42?.Equiped with portable testing instruments detection of GM mazie can be done in fields.Untill now,China has approved a series of GM maize events including 3272,59122,Bt176,Bt11,GA21,MIR162,MIR604,MON810,MON863,MON88017,MON89034,NK603,T25,TC1507.This study aim to develop a series of RPA rapid DNA detection methods for these approved GM maize events base on materials as mentioned above.The main content and result are as follows:1.Development of RPA rapid detection methods of GM maize lines.Paris of event-special detection primers and probes are designed between the cloned fragment and maize genome junction regions.By screening tests,the best primers and probes are chosen to develop the RPA fluorescence detection method.The detection methods of Bt11,Bt176,TC1507,MON810,MON863,MON89034 have been established,occupy 43% of the GM maize approved.The result has proved that these RPA methods have the high sensitivity and specificity,and can detect stably as few as 50 copies of the targets within 20 minutes.2.Development of duplex RPA detection method for GM maize TC1507 and MON89034: We choose TC1507 and MON89034 as materials and use the event-specific method as mentioned above to develop duplex RPA assay.The results show that both TC1507 and MON89034 can be detected simultaneously at mixed samples,and the sensitivity of TC1507 and MON89034 are 500 copies and 50 copies respectively.