Background In the last century, Influenza A viruses have caused the most global pandemics. The global panzootic of Highly Pathogenic Avian Influenza A (H5N1), its expanding mammalian host range, first mammalian-to-human transmissions and rising human infections are ominous. As a leading pandemic risk, early H5N1 detection is crucial to outbreak response but currently remains limited to the CDC and state public health laboratories. Here, we present a simple, rapid, adaptable, high throughput multiplex fragment analysis test for Influenza A (H5N1, H3N2, H1N1) subtyping and Influenza A/B, RSV and SARS-CoV-2 identification.

Methods From the most up to date viral genomes we designed novel primers targeting conserved pathogenic regions across clinically relevant Influenza subtypes and generated unique gene-specific subtype signatures for H5N1 Influenza, endemic Influenza strains, RSV and SARS-CoV-2 via fragment analysis by capillary electrophoresis. Analytic sensitivity was determined by limit of detection analysis of Influenza A viral RNA in replicate reactions for each input amount of the viral RNA genome. Inter-/intra-run variability was assessed for all gene targets. Analytical specificity was established in evaluations of H5 and non-H5 human and avian Influenza A virus genomic RNA and by in silico analysis of potential primer cross-reactivity with other pathogens using BLAST® suite analysis.

Results We generated unique fragment size-based signatures for each targeted gene. Targeted conserved regions in the HA gene are conserved in all newly sequenced U.S. Human H5N1 cases. The generated H5 amplicon had CT-values 2-12 lower than CDC and WHO H5 primers. No confounding false positives or over-lapping non-specific amplicons were identified. 100% H5N1 detection sensitivity was observed with a lower limit of detection of 2.5 copies/uL.

Conclusion We have applied fragment analysis to Influenza A subtyping and respiratory virus detection for the first time. The multiplex target fragment analysis test described here has 100% analytical sensitivity and no false positives. This enables total subtyping capability of all human circulating Influenza A strains in one assay. This platform also proposes an epidemiological tool for detecting possible H5 Avian strain cross-over via dual detection of the H5 and M gene without the N1 amplicon. The use of multiple gene-specific targets for each strain increases test rigor, reproducibility and confidence in the accuracy of results.