Performance Assessment of Avian Influenza Virus Analytical Reference Materials for Diagnostic Surveillance: Subtypes H5N1, H7N9, H7N7, H5N6, and H9N2

Highly pathogenic avian influenza (HPAI) and human influenza viruses pose major public health risks due to their potential for widespread illness and economic consequences. Early detection and control of outbreaks rely on effective surveillance and diagnostic testing. ATCC® developed a comprehensive suite of quantitative synthetic analytical reference materials (ARMs) for HPAI virus serotypes H5N1, H5N6, H7N7, H7N9, and H9N2; human influenza A virus serotypes H1N1, H3N2, and H1N1 2009 pandemic; and Influenza B virus strains. Each synthetic ARM contains sequences from the HA, NP, NA, M1, M2, NS1, and NEP genes, covering 50% of the influenza genome. The ARMs are manufactured using a reliable synthetic biology technology, verified through next-generation sequencing, and quantified via digital PCR. The ATCC® synthetic ARMs were experimentally evaluated using several published quantitative PCR (qPCR) assays, including those from the Centers for Disease Control and Prevention (CDC), the World Health Organization (WHO), and the World Organization for Animal Health (WOAH). Additionally, the avian influenza ARMs were confirmed to be compatible with subtype-specific loop-mediated isothermal amplification (LAMP) assays, and the BioFire Diagnostics FilmArray (bioMérieux) in vitro diagnostic device. The data resulting from our testing demonstrate that the ATCC® synthetic ARMs for avian and human influenza are effectively designed and suitable for developing and validating molecular-based detection and quantification assays. The synthetic RNA products displayed comparable performance to genomic RNA during all tests where comparison was feasible, making them valuable BSL-1 alternatives to BSL-3–derived materials. Our results indicate that these synthetic ARMs can serve as reliable and safe controls for molecular assays used in diagnostics and surveillance.