Development Of A Loop-Mediated Isothermal Amplification Method For Detection Of Genetically Modified Rice

In recent years, transgenic technology has been widely applied in modern agriculture and according to a report by ISAAA (The International Service for the Acquisition of Agri-biotech Applications), in2010genetically modified (GM) plants were cultivated on an estimated167million hectares. Since consumers concerns about the safety of GM food, a series of laws have been issued for regulation and labeling of genetically modified organisms (GMOs) in China, European Union (EU), Korea, Brazil, Japan and Australia, etc. However, the risk of contamination with unauthorized GM material is increasing. Thus, it is especially important to develop a rapid, sensitive and reliable method for the detection of GM rice and its processed products.Loop-mediated isothermal amplification (LAMP) is a novel method developed by Notomi et al.(2000) for the amplification of nucleic acids. LAMP is simpler than PCR, using only a water bath or heating block, and results of LAMP can be easily observed by the naked eye through a color change of the reaction mixture with SybrGreen I dye. However, this procedure has rarely been applied for the detection of GM foods and only few researches on the detection of GM soybean were reported.In this study, we developed a LAMP method for the detection of insect-resistant rice and its products. The reaction conditions were optimized including the concentrations of Mg2+and Bst DNA polymerase, reaction temperature and reaction time. The LAMP reaction was carried out in25μL containing20mM Tris-HCl (pH8.8),10mM KCl,10mM (NH4)2SO4,4mM MgSO4,0.1%Triton X-100,3mM MgCl2,5M betaine,2.8mM each dNTP,1.28μM each FIP and BIP,0.1μM each F3and B3,6.4U Bst DNA polymerase large fragment, and100ng template DNA, while the no template control contained water instead of the template. The mixture was incubated at61℃for60min in a heating blockand then heated to80℃for10min to terminate the reaction. The LAMP amplified DNA was analyzed by2%(w/v) agarose electrophoresis in1xTAE with ethidium bromide dye staining. Also, the product of the LAMP could be directly observed by naked eye after adding2μl1000×SybrGreen I into15μL reaction mixture after the amplification; the color of the mixture became green when there was a positive reaction while it remains orange where there was no amplification.The limit of detection of the developed LAMP method is0.005%. All the results demonstrated that the developed loop-mediated isothermal amplification method is rapid, convenient and reliable for on spot detection of GM rice and its processed products.