A universal RT-qPCR assay for "One Health" detection of influenza A viruses.
Authors: Nagy Alexander, Černíková Lenka, Kunteová Kateřina, Dirbáková Zuzana, Thomas Saumya S, Slomka Marek J, Dán Ádám, Varga Tünde, Máté Martina, Jiřincová Helena, Brown Ian H
Journal: PloS one
Summary
# Editorial Summary Influenza A virus (IAV) surveillance across species represents a critical gap in infectious disease monitoring, with different diagnostic assays currently recommended for avian, swine, human and equine populations despite the virus's documented ability to cross these species barriers. Nagy and colleagues developed a universal RT-qPCR assay targeting a highly conserved region of the viral matrix protein segment, analysing nearly 100,000 sequences to identify primers and probes suitable for detecting all known IAV subtypes regardless of host origin or geographic source. The optimised assay achieved 93–97% efficiency with a limit of detection of approximately ten viral templates per reaction, demonstrating 99% predicted sensitivity across the dataset analysed, with particular strength in human strains (100% inclusivity) and avian viruses (98.2%), though slightly lower for swine isolates (94.6%). Validation across 1,970 clinical specimens from six international veterinary and human diagnostic laboratories confirmed these theoretical predictions, establishing the assay's repeatability and robustness. For equine professionals, this work offers a genuinely integrated diagnostic tool aligned with One Health principles—enabling more consistent cross-species surveillance, reducing the need for multiple assays, and potentially improving early detection of novel IAV variants threatening horses and the wider animal–human interface.
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Practical Takeaways
- •This universal assay enables standardised influenza A detection across equine, avian, and swine populations using a single test, improving disease surveillance and outbreak response.
- •The high sensitivity (LOD ~10 templates) and broad host coverage make this tool practical for on-farm or clinic screening before confirmatory testing, supporting rapid risk assessment.
- •Implementation of this assay supports One Health biosecurity protocols by allowing consistent detection standards across multiple species, facilitating earlier identification of zoonotic risks and cross-species transmission events.
Key Findings
- •A universal RT-qPCR assay was developed targeting the conserved matrix protein segment of influenza A viruses with 93-97% efficiency and LOD95% of approximately 10 templates per reaction.
- •In silico evaluation showed 99% predicted detection rate across all IAV strains with 100% sequence match for human H3N2 and pandemic H1N1.
- •Validation across 1970 specimens (1455 clinical) from six international laboratories confirmed the assay's high repeatability, reproducibility, and robustness across diverse IAV strains from multiple host species.
- •The assay demonstrated perfect inclusivity for equine, avian, swine and human influenza viruses, enabling unified One Health monitoring across different animal hosts and humans.