The Establishment Of A Specific Detection Method For The PA110-15 Strain Of Transgenic Rice

With the global GM products increasingly increase,and more and more GM crops specific strains obtained in commercial production,event-specific detection has become the development trend of the international Genetically Modified Organisms.For the detection of exogenous transgenic ingredients of genetically modified(GM)food,the detection tend to not only for the conventional single target nucleic acid detection but also for rapid,accurate and high-throughput multiple targets detection analysis at the same time.There have been 4 kinds of nucleic acid detection methods based on the specificity,screen-specificity detection,gene-specificity detection,construct-specificity detection and event-specificity detection,which increasingly enhanced in the specific test.Currently,event-specific detection approaches always contain conventional PCR,real-time PCR and LAMP.Respectively,they have their own advantages and disadvantages.PA 110-15 is a kind of unauthorized genetically modified rice,which storage high lysine protein genes transferred by agrobacterium mediated.In order to better monitor the existence of genetically modified rice event PA110-15 and meet the requirements of the management of GM identification,this study has established 3 novel event-specific detection methods for PA110-15 with normal PCR,quantitative real-time PCR and LAMP..In the long term,the establishment of specific detection approaches of GM rice PA 110-15 can effectively prevent the unauthorized genetically modified rice spreading to the market,and promote the exportation of Chinese rice and products.Most important of all,safety evaluation through screening and monitoring of transgenic plant would be enhanced through establishing specific detection of GM rice PA110-15.About DNA extraction,SDS method was applied for PCR and LAMP detection,while DNA for real time fluorescent PCR detection was extracted by magnetic bead adsorption,because high concentration of DNA template was available by this method,and DNA quality is good.In this study,three novel approaches have been developed based on 5' flanking sequence of genetically modified rice PA 110-15.Firstly,conventional PCR method was established for the event-specific detection of GM rice PA110-15;secondly,event-specific quantitative real-time PCR method was developed for the detection of GM rice PA110-15;Thirdly,LAMP method was developed for the event-specific detection of GM rice PA110-15.Experimental results proved that all these three established methods are specific and stable.The sensitivity detection of LAMP menthod is higher than RT-PCR and conventional PCR,which showed 5 copies?L and 20 copies/?L respectively.However,among three developed methods,LAMP is easy to contaminate during the experiment.Above all,all these 3 methods meet the routine analysis requirements for GM rice PA110-15,and the specific methods can be selected according to the specific circumstances.