Abstract
Background:
Newcastle disease (ND) is a highly contagious and often fatal viral disease that affects a wide range of avian species, regardless of age or sex. It continues to represent a significant challenge to the productivity and survival of both commercial and traditional poultry, particularly in developing countries. Despite advancements in vaccination strategies, ND outbreaks remain a recurring issue in many areas, including Morocco. Early detection of NDV is critical for effective disease management, but conventional virus isolation methods are labor-intensive and time-consuming.

Aim:
This study aimed to evaluate and validate the performance of the ID Gene™ Newcastle Disease Duplex realtime RT-PCR kit for the rapid and sensitive detection of NDV in poultry samples. The validation was performed in accordance with international OIE standards to ensure reliability and applicability in veterinary diagnostics.

Methods:
The study utilized spiked biological samples and NDV strains, including Moroccan field strains and reference strains from the Isntituto Zooprofilattico Sperimentale delle Venezie proficiency panel. RNA extraction was performed using the NucleoSpin® RNA Virus kit. Negative tracheal and cloacal swab supernatants, as well as homogenized tissue samples from the liver and lungs, were spiked with JEL/Morocco Newcastle virus, genotype VI
at different concentrations. Real-time RT-PCR targeting the NDV M gene was performed with an exogenous internal control for duplex amplification. The analytical specificity was assessed for inclusivity with various NDV genotypes and exclusivity against other orthomyxoviruses. Sensitivity was determined using 10-fold serial dilutions of NDV, with detection limits evaluated for individual and pooled samples. Repeatability was assessed by calculating intraassay and inter-assay coefficients of variation based on Ct values.

Results:
The ID Gene™ kit demonstrated excellent inclusivity and reliably detected all tested NDV strains irrespective of genotype while showing no cross-reactivity with non-NDV orthomyxoviruses. The analytical sensitivity was validated at 102 DIO50/ml for individual samples and 103 DIO50/ml for pools of five samples, respectively, across all tested matrices. The intraassay and interassay repeatability coefficients of variation (CV) were consistently below 10%, confirming the robustness of the method.

Conclusion:
The ID Gene™ Newcastle Disease Duplex Kit is a rapid, accurate, and sensitive diagnostic alternative to traditional virus isolation methods. The performance of this method, validated against international and national standards, highlights its potential as a reliable tool for the early detection and effective management of NDV infections in Moroccan poultry populations.

Key words: Avian orthoavulavirus type I, Polymerase chain reaction (PCR), Validation