Abstract
Newcastle Disease Virus (NDV) is a major avian pathogen causing significant economic losses worldwide. Rapid and accurate detection is essential for effective outbreak control and epidemiological surveillance. In this study, SYBR Green-based qPCR primers targeting the M and F genes of NDV were designed, optimized, and evaluated. Selected primers amplified NDV strains (LaSota and Almaty03) with no cross-reactivity observed for other avian viruses, including Avian influenza (AIV) and Fowlpox virus (FWPV), confirming high specificity. Sensitivity testing with plasmid DNA showed initial copy numbers of 3.03 × 10⁹ copies/µL (F gene) and 3.30 × 10⁹ copies/µL (M gene). Ten-fold serial dilutions (10⁻1–10⁻⁵) revealed a limit of detection of 3 × 10⁴ copies/µL for both genes. Melt curve analysis confirmed single, specific peaks without non-specific amplification. These results demonstrate that the optimized primer sets provide sensitive and specific detection and reliable for NDV detection. Incorporation of sequence-specific probes in future studies may further improve sensitivity and quantitative accuracy, supporting broad applications in diagnostics and molecular research.