Fluorometric detection of influenza virus RNA by PCR-coupled rolling circle amplification generating G-quadruplex

Abstract We developed a label-free fluorometric system based on double amplification of target nucleic acid by rolling circle amplification generating G-quadruplex (GQ-RCA) for sensitive detection of influenza virus RNA. To detect viral RNA with high sensitivity and selectivity, double amplification system was employed through reverse transcription PCR (RT-PCR) coupled in tandem with GQ-RCA. Single-stranded amplified viral DNA (ssDNA) was generated by RT-PCR of samples containing influenza virus RNA. The phosphate-primed DNA strand in amplicon DNA was subsequently degraded by lambda exonuclease. Viral ssDNA formed a ternary RCA initiation complex by annealing to both a partial hairpin primer and a dumbbell padlock DNA template. A long stretch of ssDNA containing repeated copies of the G-quadruplex sequence was generated by RCA at room temperature. Sensitive detection of amplified target nucleic acid was then accomplished by monitoring fluorogenic interactions between Thioflavin T (ThT) and RCA-responsive G-quadruplexes. Differences in fluorescence intensity between target and non-target viral RNA were evident under UV illumination. Selective fluorescence staining of RCA-responsive G-quadruplexes enabled influenza virus RNA detection at concentrations as low as 4.9 aM with a linear detection range between 450 aM and 450 fM. The RT-PCR-coupled GQ-RCA system for influenza virus genome detection exhibited very high sensitivity and allowed convenient multiplexed virus detection within 2.5 h. Thus, a combination of RT-PCR-coupled GQ-RCA and ThT fluorescence staining can be a sensitive and accurate method for detecting RNA molecules of influenza viruses as well as those of other viruses.