Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.).

摘要

The cry1Ab gene is a foreign gene which encodes Bt insecticidal Cry1Ab protein and was transferred into genomic DNA of plants to acquire insect resistance. Here a loop-mediated isothermal amplification (LAMP) assay with high specificity and rapidity under isothermal conditions was developed for detecting cry1Ab gene in transgenic rice. Partial sequence of cry1Ab gene was used as the target template to design LAMP primers. The reaction conditions were optimized as follows: 60 min of reaction time, 1:3 of outer primer and inner primer concentration ratio, 25 muL of reaction volume and 0.6 muM of betaine. The results of detection could be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with that of regular PCR and real-time PCR. The results showed that the LAMP assay exhibited high specificity and the sensitivity of 3 x 102 copies number of the positive control plasmid, and of 0.5% genetically modified (GM) contents. In comparison with real-time PCR method, LAMP showed the same results with simple instruments. The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for detecting cry1Ab gene from transgenic rice plants and rice ingredients in processed foods aimed at screening the growing transgenic crops in the fields and detecting genetically modified (GM) ingredients in imported and domestic foods