Development of a universal real-time PCR TaqMan assay for fowl aviadenovirus diagnosis

Fowl aviadenoviruses (FAdVs) are major pathogens of poultry, associated with economically important diseases such as Inclusion Body Hepatitis (IBH), Adenovirus Gizzard Erosion (AGE), and Hepatitis-Hydropericardium Syndrome (HHS). Accurate diagnosis is critical for disease control and surveillance, yet current methods are limited by serotype diversity. This study describes the development and validation of a universal TaqMan real-time PCR assay for the detection and quantification of multiple FAdV serotypes. Universal primers and a probe were designed to target a conserved region of the 52K gene and optimized for assay performance. Analytical evaluation demonstrated high efficiency (95 %), strong linearity (R² = 0.955), and a detection limit of 5 copies/mL. The assay showed no cross-reactivity with other avian pathogens, including avian influenza virus, Newcastle disease virus, infectious bronchitis virus, and infectious laryngotracheitis virus. Precision testing revealed minimal intra- and inter-assay variability, with coefficients of variation below 0.02 %, confirming robustness and reproducibility. Clinical evaluation was conducted on 56 field samples from confirmed IBH and AGE cases involving serotypes FAdV-1, -8a, -8b, and -11 as well as 14 samples from healthy chicken. The assay successfully amplified all positive samples and showed a strong correlation (R² = 0.97) with a previously established SYBR Green-based 52K real-time PCR assay. In conclusion, this novel TaqMan real-time PCR assay represents a rapid, sensitive, and highly specific diagnostic tool for FAdV detection. Its broad applicability across serotypes and strong clinical performance make it suitable for both routine diagnostics and epidemiological surveillance of FAdV infections in poultry.