The Application Of Recombinase Polymerase Amplification(RPA) Technology In Detection Of Transgenic Rices

The commercial cultivation area of genetically modified organisms(GMOs)has increased more than 100 times since 1996.The species of transgenic crops also increased year by year.As for biosafety of GMOs,the public concerned more and more,meanwhile,the regulation and monitoring strength are keeping rising in many countries.Transgenic rice researches are very active in China.As one of the most important staple food for most Chinese,the public acceptance of GM rices is still rather low.To satisfy the public’s right to know and government regulation,detection technology of transgenic crops has been a research hotspot in GM biosafety research.Thus,it is necessary to develop a fast,sensitive and accurate detection method for GMOs.Traditional PCR cannot meet the demands of field test.The advent of the isothermal amplification technology can meet the demands.In this study,we chose the recombinase polymerase amplification(RPA)for its various excellent advantages.RPA is a fast and sensitive isothermal amplification technology,which reaction temperature is closer to room temperature than other technologies.It’s reaction temperature is between 25~42℃,and its reaction time is less than 20 min.We developed RPA detection methods for four kinds of transgenic rices lines which are developed in China and have the potential to commercialize.According to the flanking sequences and the exogenous gene sequences of GM rice line mf-MH3301-1,we screened out a pair of primers and a probe suited for RPA detection.Then we established a fast detection method include RPA-Basic and RPA-EXO.RPA-EXO method can stably detect GM mf-MH3301-1 with 100 copies.According to the flanking sequences and the exogenous gene sequences of GM rice lines PA110-15 and Bar68-1,we screened out primers pairs and probes suited for RPA detection respectively.Then we established a fast detection method include RPA-Basic,RPA-EXO and RPA-LFD.RPA-EXO method were constructed with detection limet of 500 copies and 50 copies for detecting GM lines PA110-15 and Bar68-1 repectively.RPA-LFD method were constructed with detection limet of 75 copies and 50 copies for detecting GM lines PA110-15 and Bar68-1 repectively.For KMD1,accroding to its 5’ flanking sequence of the exogenous gene,we screened out several pairs of primers.And a probe is suitable for specificity detection.In conclusion,we discussed the disadvantages and advantages of RPA in the qualitative and quantitative detection of transgenic rices,provided a research base for the rapid detectionof GM rices in fields.