Development of Human and Avian Influenza Analytical Reference Materials for Surveillance and Diagnostics

Background 

Human and highly pathogenic avian influenza (HPAI) viruses pose a significant public health threat, with the potential to cause widespread illness and economic losses. Early detection and control of outbreaks depend on effective surveillance and diagnostic testing. 

Method 

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 the complete sequences from segments 4, 5, 6, 7, and 8, including the HA, NP, NA, M1, M2, NS1, and NEP/NS1 genes, covering 50% of the influenza genome. These segments are key diagnostic targets for molecular tests and provide sufficient genomic context for assessing assay specificity. These ARMs are manufactured using a highly reliable synthetic biology technology, verified by next-generation sequencing, and quantified via digital-based PCR. Further, they do not contain any viable material and can be handled in BSL-1 settings. As such, they are intended to serve as safe and reliable positive controls for molecular tests for surveillance and diagnostics. 

Results 

These 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. We conducted an in silico assessment of ARM compatibility using over 250 published assays. The synthetic products performed as well as genomic RNA in all tests. 

Conclusions 

The data show that all synthetic ARMs for avian and human influenza are effectively designed and suitable for developing and validating molecular detection and quantification assays. Moreover, our findings indicate that the synthetic RNA ARMs are equivalent to their corresponding genomic RNA, making them a valuable BSL-1 alternative to BSL-3–derived materials. Our results suggest that these synthetic ARMs can serve as reliable and safe controls for molecular assays used in diagnostics and surveillance.