Development of Fit-for-Purpose, High Quality Proficiency Samples for Interlaboratory Evaluation of RT-PCR Detection of HPAI H5N1 in Milk

The increased demand for highly pathogenic avian influenza (HPAI) testing in milk highlights the critical need for well-characterized samples that enable meaningful evaluation of current detection methods. In this study, we focus on the development and rigorous characterization of artificially contaminated spiked milk samples, forming the foundation for reliable assessment of assay performance. Both real-time reverse transcriptase PCR (rRT-PCR) and reverse transcriptase digital PCR (RT-dPCR) are viable methods to detect HPAI. Though standard curves can be used in rRT-PCR assays to estimate viral genome copy (GC) numbers in samples, RT-dPCR measurements provide absolute quantification of sample GC numbers. By using RT-dPCR, we obtained precise GC measurements of our virus stock and of spiked samples, producing accurate quantification even at low viral loads. In parallel, the U.S. Department of Agriculture National Veterinary Services Laboratories’ (USDA-NVSL’s) influenza A matrix gene (IAV M) rRT-PCR assay was employed as the main detection assay, serving as a tool to validate the sample preparation methodology by evaluating both the consistency of detection across GC levels and the stability of samples over time. The goal of this work is to show that robust, reproducible, and analytically traceable samples can be generated to support HPAI method evaluation in proficiency exercises (PEs) distributed nationwide. This study defines the assay sensitivity, expressed as the level of detection 50% (LOD _50% ), of the USDA-NVSL’s IAV M rRT-PCR assay, setting the basis for determining laboratory competency and providing confidence in HPAI testing results for ongoing and future surveillance.