Fluorescence nucleobase analogue-based strategy with high signal-to-noise ratio for ultrasensitive detection of food poisoning bacteria

Abstract

We developed a simple and ultrasensitive strategy for the identification of foodborne pathogens utilizing a fluorescent nucleobase analogue [2-aminopurine (2-AP)]-containing split G-quadruplex that binds blocker DNA. Compared to a previous strategy that did not use blocker DNA, this strategy showed a significant increase in the signal-to-noise ratio—by approximately 300%—owing to the displacement of the blocker DNA by the target DNA that induces the formation of an active G-quadruplex structure, thereby leading to a substantial increase in the 2-AP fluorescence signal. The proposed strategy was rationally combined with polymerase chain reaction, which resulted in the successful determination of genomic DNA (within the range of 10–10⁶ copies) derived from the food poisoning bacterium Escherichia coli, with a limit of detection of 5.2 copies and high selectivity. In addition, the practical applicability of this method was demonstrated by analyzing E. coli-spiked lettuce samples.