Label-free fluorometric detection of influenza viral RNA by strand displacement coupled with rolling circle amplification

Abstract

Since influenza occurs globally every year, it is important to develop a facile and accurate method to detect the influenza virus. This study aimed at developing a sensitive fluorometric assay for detecting influenza viral RNA through tandem gene amplification methods including reverse transcription PCR (RT-PCR), followed by strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). Influenza viral RNA was initially amplified by RT-PCR with a tailed reverse primer containing an additional sequence for SDA. The RT-PCR amplicon was then subjected to SDA, yielding multiple copies of single-stranded DNA (ssDNA) that can be used as a primer for subsequent RCA. Thereafter, a long ssDNA segment harboring tandem repeated G-quadruplexes that were generated through RCA was intercalated by Thioflavin T, yielding a strong fluorescence signal indicating the presence of the target viral RNA. Fluorometric analysis detected influenza viral RNA ranging from 50 pg to 500 pg with a limit of detection of 6.2 pg with a signal-to-background ratio of 10 and identified each influenza virus strain (H1N1, H3N2, and influenza B). Thus, the present method for the label-free fluorometric detection of viral RNA via tandem gene amplifications combining RT-PCR-coupled SDA and G-quadruplex-generating RCA would facilitate the efficient diagnosis of influenza infection.