Evaluation of Clinical, Analytical, and Genotyping Performance of Hex L1 PCR Coupled with High-Resolution Melting Curve Analysis for Outbreak Investigation in Morocco.

Fowl adenoviruses (FAdVs) are widespread viruses in poultry populations, responsible for several severe diseases, including Inclusion Body Hepatitis (IBH), Adenoviral Gizzard Erosion (AGE), and Hepatitis-Hydropericardium Syndrome (HHP). These diseases have significant economic and health impacts on poultry industries globally. In this study, we aimed to evaluate the clinical, analytical, and genotyping performance of the Hex L1 PCR combined with High-Resolution Melting (HRM) Curve analysis for investigating recent IBH and AGE outbreaks in Morocco. The study involved 26 clinical samples collected from broiler and layer poultry farms suspected with IBH and AGE. These samples were amplified using conventional PCR, real-time PCR/52K test, and the Hex L1 PCR/HRM test. Field isolates were also sequenced and compared with HRM curve analysis results to validate the genotyping accuracy of the Hex L1 PCR/HRM method. Phylogenetic analysis of the sequenced samples revealed several FAdV genotypes, including FAdV-11 and FAdV-8b in IBH cases, and FAdV-1 and FAdV-8a in AGE cases, highlighting the genetic diversity of circulating strains. The Hex L1 PCR/HRM method successfully amplified all 12 FAdV serotypes, demonstrating excellent reproducibility and repeatability, with coefficients of variation ranging from 0.19% to 1.82%. Moreover, this method showed a strong correlation with the real-time PCR/52K method, achieving a high correlation coefficient of 0.9077. The HRM curve analysis reliably genotyped all the field isolates, and the results matched exactly with the sequencing outcomes. In conclusion, this method offers a fast, sensitive, and reliable alternative for FAdV detection and genotyping. It provides universal detection, quantification, and genotyping in a single step, overcoming the limitations of traditional techniques, making it a perfect tool for epidemiological studies and outbreak investigation.