Zhou Dinggang, Guo Jinlong, Xu Liping, Gao Shiwu, Lin Qingliang, Wu Qibin, Wu Luguang, Que Youxiong
Key Laboratory of Sugarcane Biology and Genetic Breeding, Fujian Agriculture and Forestry University, Ministry of Agriculture, Fuzhou 350002, China.
School of Agriculture and Food Sciences, The University of Queensland, Brisbane 4072, Australia.
Sci Rep. 2014 May 9;4:4912. doi: 10.1038/srep04912.
To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg(2+), 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 μL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn(2+) (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane.
为满足基因技术监管中对转基因甘蔗中外源基因检测的需求，基于环介导等温扩增（LAMP）技术，针对cry1Ac基因开发了一种高效、高特异性且快速的检测方法。利用cry1Ac序列设计了一组四条引物，并优化了反应条件：在25.0 μL反应体积中，Mg(2+)浓度为5.25 mM，内引物与外引物比例为4:1，Bst DNA聚合酶用量为2.0 U。结果表明，三种LAMP后检测方法（沉淀法、0.60 mM钙黄绿素与0.05 mM Mn(2+)复合物法以及SYBR Green I可视化法）均有效。当使用重组cry1Ac质粒或cry1Ac转基因甘蔗植物基因组DNA作为模板时，LAMP方法的灵敏度比传统PCR高十倍。更重要的是，该系统无需复杂且昂贵的仪器，仅凭肉眼就能在筛选转基因甘蔗时现场检测外源基因。此方法不仅可为cry1Ac检测提供技术支持，还能进一步推动该检测技术用于转基因甘蔗中其他转基因的检测。
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