Li Feiwu, Yan Wei, Long Likun, Qi Xing, Li Congcong, Zhang Shihong
College of Plant Sciences, Jilin University, Changchun 130062, China.
Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences, Changchun 130033, China.
Int J Mol Sci. 2014 Aug 27;15(9):15109-21. doi: 10.3390/ijms150915109.
The cry2Ab and cry3A genes are two of the most important insect-resistant exogenous genes and had been widely used in genetically-modified crops. To develop more effective alternatives for the quick identification of genetically-modified organisms (GMOs) containing these genes, a rapid and visual loop-mediated isothermal amplification (LAMP) method to detect the cry2Ab and cry3A genes is described in this study. The LAMP assay can be finished within 60 min at an isothermal condition of 63 °C. The derived LAMP products can be obtained by a real-time turbidimeter via monitoring the white turbidity or directly observed by the naked eye through adding SYBR Green I dye. The specificity of the LAMP assay was determined by analyzing thirteen insect-resistant genetically-modified (GM) crop events with different Bt genes. Furthermore, the sensitivity of the LAMP assay was evaluated by diluting the template genomic DNA. Results showed that the limit of detection of the established LAMP assays was approximately five copies of haploid genomic DNA, about five-fold greater than that of conventional PCR assays. All of the results indicated that this established rapid and visual LAMP assay was quick, accurate and cost effective, with high specificity and sensitivity. In addition, this method does not need specific expensive instruments or facilities, which can provide a simpler and quicker approach to detecting the cry2Ab and cry3A genes in GM crops, especially for on-site, large-scale test purposes in the field.
cry2Ab和cry3A基因是两个最重要的抗虫外源基因，已被广泛应用于转基因作物中。为了开发更有效的方法来快速鉴定含有这些基因的转基因生物（GMO），本研究描述了一种用于检测cry2Ab和cry3A基因的快速可视化环介导等温扩增（LAMP）方法。LAMP检测可在63℃等温条件下60分钟内完成。通过实时浊度仪监测白色浑浊度可获得LAMP产物，或者通过添加SYBR Green I染料直接肉眼观察获得。通过分析13种具有不同Bt基因的抗虫转基因作物事件来确定LAMP检测的特异性。此外，通过稀释模板基因组DNA来评估LAMP检测的灵敏度。结果表明，所建立的LAMP检测的检测限约为单倍体基因组DNA的5个拷贝，比传统PCR检测高约5倍。所有结果表明，所建立的这种快速可视化LAMP检测快速、准确且具有成本效益，具有高特异性和灵敏度。此外，该方法不需要特定的昂贵仪器或设备，可为检测转基因作物中的cry2Ab和cry3A基因提供一种更简单、更快速的方法，特别是用于现场大规模田间检测目的。
