Development of a Multiplex Conventional PCR Assay for Concurrent Detection of FAdV-4, FAdV-8b, and FAdV-11

Fowl adenovirus (FAdV) belongs to the Aviadenovirus genus within the Adenoviridae family. FAdVs are widely distributed and associated with various diseases in poultry, including adenoviral gizzard erosion (AGE), hepatitis-hydropericardium syndrome (HHS), and inclusion body hepatitis (IBH). In this study, we developed a multiplex conventional PCR for simultaneously detecting FAdV-4, -8b, and -11 by targeting the hexon gene. The multiplex PCR was optimized for primer concentrations and thermocycling conditions. The optimal primer concentration combination was set at 0.125 μM for FAdV-4, -8b, and 0.25 μM for FAdV-11. Under these conditions, the limit of detection (LOD) was 10³ copies/μL of plasmid standards for FAdV-4, -8b, and -11. These results demonstrated that the developed multiplex PCR method exhibits high specificity and sensitivity, with no observed cross-reactivity among these serotypes or with other poultry viruses. Therefore, this multiplex PCR will be an effective tool for accurate serotyping of FAdV-4, -8b, and -11, enabling more precise identification and differentiation of these three serotypes.