Analysis of genetically modified soya and soya products using a fluorescence probe based on PCR-initiated isothermal amplification of G-quadruplex DNA

Abstract

We proposed a novel strategy for the analysis of genetically modified (GM) soya samples by combining PCR-initiated generation and isothermal amplification of G-quadruplex DNA. PCR primers were designed to consist of a target-specific sequence, a G-quadruplex complement sequence, and a nicking endonuclease recognition site. After the PCR step, the nicking enzyme cut the recognition site, and single stranded G-quadruplex sequences were displaced and further amplified by polymerase during the isothermal amplification reaction. Then, the G-quadruplex sequences combined with N-methyl mesoporphyrin IX to generate strong fluorescence. This method has a detection limit of 0.2% and a linear range from 50% to 0.5% GM content. When exponential isothermal amplification was coupled to the preceding amplification step, the detection limit was further improved to 0.02% GM soya. GM contents were successfully detected at 0.1% GM percentage in soy milk and 0.9% GM percentage in tofu and dried tofu samples. The method may be applied to the analysis of various GM species simply by changing the target sequence in the primers.