Abstract
Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention’s (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC’s probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.
Highlights
Influenza is a rapidly evolving viral pathogen that infects up to 5 million people annually [1]
We showed that Fast Evaluation of Viral Emerging Risks (FEVER) probes are compatible with a flow cytometer sensing strategy and could be applied with minimal difficulty to the diagnostic field [47]
The IAV and IBV FEVER probes are predicted to detect influenza RNA with a high mismatch tolerance needed for future biosurveillance and diagnostic applications
Summary
Influenza is a rapidly evolving viral pathogen that infects up to 5 million people annually [1]. Pandemics occur when a new viral strain evolves through genome reassortment producing influenza variants for which there is no pre-existing human immunity [4,5,6]. These novel variants often emerge from bird or pig reservoirs [7]. A method for rapid and accurate point-of-care viral diagnostics, both for influenza and other pathogens, would provide time to implement prevention measures and to rule out pandemic infections so that patients receive proper medical treatment [9]
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