Development and validation of a sensitive fluorescence RT-qPCR assay with TaqMan-MGB probe for detection of H6 subtype avian influenza A virus

Avian influenza, caused by the avian influenza A virus (IAV), threatens poultry and public health. H6 subtype avian IAV is a low-pathogenic virus with hosts ranging from poultry and wild birds to mammals. H6 persists latently in poultry, which enables silent transmission and cross-species risk. The few fluorescence quantification assays that exist for H6 are mostly multiplexed. We developed a rapid, sensitive, efficient, monoplex fluorescence reverse-transcription qPCR (RT-qPCR) assay for H6 IAV. Specific primers and a TaqMan-MGB probe were designed based on the conserved hemagglutinin (HA) gene region of H6 IAVs from the GISAID database. The reaction components and conditions were optimized, and the assay was evaluated for specificity, sensitivity, and reproducibility. The optimized assay had excellent specificity, with no cross-reactivity with other avian viruses, including IAV subtypes H1-5, H7, H9, and H10, Newcastle disease virus, infectious bronchitis virus, fowl adenovirus, infectious laryngotracheitis virus, chicken anemia virus, <i>Mycoplasmopsis</i> (<i>Mycoplasma</i>) <i>gallisepticum</i>, and <i>M. synoviae</i>. Our method had a detection limit of 8.2 × 10⁰ copies/μL, which is 1,000 times more sensitive than conventional RT-PCR. The intra- and inter-assay CVs for all tested concentrations were both <1.5%, indicating good reproducibility. When applied to clinical swab samples, the sensitivity of our fluorescence RT-qPCR assay was 98.8% and specificity was 96.2% compared with traditional virus isolation. Our method could provide strong technical support for the early detection, monitoring, and prevention of H6 subtype IAV infection.