Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane

摘要

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 Mg2+, 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 Mn2+ (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.