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 Šand 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.

Recognition of dual targets by a molecular beacon-based sensor: subtyping of influenza A virus.

A molecular beacon (MB)-based sensor to offer a decisive answer in combination with information originated from dual-target inputs is designed. The system harnesses an assistant strand and thermodynamically favored designation of unpaired nucleotides (UNs) to process the binary targets in "AND-gate" format and report fluorescence in "off-on" mechanism via a formation of a DNA four-way junction (4WJ). By manipulating composition of the UNs, the dynamic fluorescence difference between the binary targets-coexisting circumstance and any other scenario was maximized. Characteristic equilibrium constant (K), change of entropy (ΔS), and association rate constant (k) between the association ("on") and dissociation ("off") states of the 4WJ were evaluated to understand unfolding behavior of MB in connection to its sensing capability. Favorable MB and UNs were furthermore designed toward analysis of genuine genetic sequences of hemagglutinin (HA) and neuraminidase (NA) in an influenza A H5N2 isolate. The MB-based sensor was demonstrated to yield a linear calibration range from 1.2 to 240 nM and detection limit of 120 pM. Furthermore, high-fidelity subtyping of influenza virus was implemented in a sample of unpurified amplicons. The strategy opens an alternative avenue of MB-based sensors for dual targets toward applications in clinical diagnosis.