Three-Way Junction-Induced Isothermal Amplification with High Signal-to-Background Ratio for Detection of Pathogenic Bacteria

Abstract

The consumption of water and food contaminated by pathogens is a major cause of numerous diseases and deaths globally. To control pathogen contamination and reduce the risk of illness, a system is required that can quickly detect and monitor target pathogens. We developed a simple and reproducible strategy, termed three-way junction (3WJ)-induced transcription amplification, to detect target nucleic acids by rationally combining 3WJ-induced isothermal amplification with a light-up RNA aptamer. In principle, the presence of the target nucleic acid generates a large number of light-up RNA aptamers (Spinach aptamers) through strand displacement and transcription amplification for 2 h at 37 °C. The resulting Spinach RNA aptamers specifically bind to fluorogens such as 3,5-difluoro-4-hydroxybenzylidene imidazolinone and emit a highly enhanced fluorescence signal, which is clearly distinguished from the signal emitted in the absence of the target nucleic acid. With the proposed strategy, concentrations of target nucleic acids selected from the genome of Salmonellaenterica serovar Typhi (S. Typhi) were quantitatively determined with high selectivity. In addition, the practical applicability of the method was demonstrated by performing spike-and-recovery experiments with S. Typhi in human serum.