Visual signal generation for the detection of influenza viruses by duplex recombinase polymerase amplification with lateral flow dipsticks

We present a detailed study on visual detection of influenza viruses by duplex recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFDs). The LFD consisted of two test lines and a control line, on which anti-fluorescein isothiocyanate antibodies, anti-digoxigenin antibodies, and biotinylated bovine serum albumin were immobilized, respectively. The performance of the LFD was evaluated with dual-labeled DNA amplicons. The results indicate that the detection of DNA amplicons by LFDs is specific and sensitive, with detection limits of 5.80 fmol for fluorescein isothiocyanate-labeled amplicons and 8.39 fmol for digoxigenin-labeled amplicons. We next developed a duplex RPA–LFD assay for simultaneous detection of influenza A virus and influenza B virus, and then optimized the parameters, including the reaction temperature, reaction time, and concentrations of primers and probes. Assessment of the specificity and sensitivity indicated that this assay is sensitive and specific for simultaneous detection of influenza viruses, with detection limits of 50 copies per reaction for influenza B virus and 500 copies per reaction for influenza A virus, without cross-reactivity with other pathogens. Compared with real-time PCR as a reference method to detect influenza viruses in clinical samples, the clinical sensitivity of the duplex RPA–LFD assay was 78.57% for influenza A virus and 87.50% for influenza B virus, with 100% specificity. In conclusion, the duplex RPA–LFD assay is a rapid, cost-effective, and sensitive method for the identification of influenza viruses.