Development of the One-step Visual Loop-Mediated Isothermal Amplification Assay for Genetically Modified Rice Event TT51-1

Abstract

Loop-mediated isothermal amplification (LAMP), a novel DNA amplification technique, is widely used inmolecular diagnosis although it is very easy to be contaminated by the huge amount of its own amplicons. An improved visual LAMP method for the event specific identification of GM rice TT51-1 and a rice endogenous reference gene (sucrose phosphate synthase, SPS) was established. Pre-loaded calcein and manganous ion into the LAMP reaction offered not only an easy way to read results by colour change from orange to green but also a closedtube system to reduce contaminations caused by amplification products. The LAMP assays could be finished within 60 min, and the limits of detection (LODs) were estimated as low as 10 and 20 copies of rice haploid genomic DNA, respectively, about 10-fold more sensitive than that of conventional PCR. The specificities of the assays were also well evaluated with optimized system and conditions. The developed one-step visual LAMP assays in this study provided a promising tool for the convenient and effective detection of GM crops.