Polymerase-assisted fluorescence resonance energy transfer (FRET) assay for simultaneous detection of binary viral sequences.

The analysis of a specific sequence of nucleic acids enables identification of pathogens and the diagnosis of human genetic disorders. This emphasises the need to develop methods of detecting nucleic acids, particularly in a multiplex format, that yield a decisive conclusion for clinical interpretation. Herein, we introduce a polymerase-assisted fluorescence resonance energy transfer (FRET) assay to simultaneously analyse binary viral genes that are characteristic of hemagglutinin and neuraminidase in influenza A virus. The approach takes advantage of the formation of an X-shaped four-way DNA junction (4WJ), thus enabling a selective response to the binary targets of specific sequences. Polymerase drives the cycling of the target complex and incorporates 2'-deoxyuridine-5-triphosphate (dUTP) labelled with Texas Red (TR-dUTP) into the double-stranded DNA (dsDNA) that is produced, which induces FRET to produce a sensing output. Crucially, the mechanism relies on a DNA hairpin instead of a molecular beacon, which substantially increases the simplicity of the assay and reduces its cost. The results revealed that the lowest detectable concentration was approximately 2 pM. The donor-acceptor distance was approximately 7 Å and independent of the ratio of TR-dUTP to 2'-deoxythymidine-5-triphosphate (dTTP). An 'off-on' assay operating in AND gate mode was demonstrated and is potentially useful for the fast diagnosis and subtyping of influenza viruses.