Development of the First specific Real-Time PCR TaqMan Assay for Fowl Adenovirus serotype 11 diagnosis.

AbstractFowl adenovirus serotype 11 (FAdV-11) is a major pathogen in poultry, associated with inclusion body hepatitis (IBH) and causing significant economic losses in the poultry industry worldwide. This study presents the development and validation of the first specific real-time PCR test based on the TaqMan probe, specifically designed for the detection, quantification, and genotyping of FAdV-11 in one reaction. Primers and a probe targeting a region within the FAdV-11 Fiber gene were designed using Geneious Prime. Validation steps, including assessments of sensitivity, linearity, reproducibility, repeatability, and specificity, were conducted using reference strains to evaluate the analytical performance of our assay. The test demonstrates excellent linearity (R² = 0.995) and analytical sensitivity of 10 copies/µl, with specific amplification of FAdV-11 without cross-reactivity with other FAdV serotypes or other avian pathogens, delivering results in only 31 min. The test shows low inter-assay and intra-assay variability, with coefficients of variation below 0.38% and 0.25%, respectively. The clinical performance of the test was validated on field samples collected from IBH clinical cases, showing perfect concordance with sequencing results. In addition, the sensitivity of our test was ten times greater than that of the cPCR test. Furthermore, a comparison of the specific real-time TaqMan PCR test with the universal real-time PCR/52 K test showed a high correlation (R2 = 0.98), confirming a good analytical performance of our test. By combining the detection, quantification, and genotyping of FAdV-11 in one reaction, this test offers a complete tool for an effective response to FAdV-11 epidemics in the poultry industry.