Development and Analytical Validation of a Multiplex Real-Time RT-PCR Assay for Simultaneous Detection of Avian Influenza A Virus and Newcastle Disease Virus.

Avian influenza A virus (AIV) and Newcastle disease virus (NDV) are among the most damaging pathogens of poultry, and AIV also carries zoonotic potential that places it within the One Health agenda. The two infections frequently produce overlapping clinical signs, so a method that detects both in a single reaction has clear practical value for surveillance. We developed a duplex one-step real-time reverse-transcription PCR (RT-qPCR) that detects AIV in the FAM channel and NDV in the ROX channel. Primers and hydrolysis probes were designed against conserved regions of the AIV nucleoprotein and matrix genes and the NDV matrix, phosphoprotein and nucleoprotein genes, after alignment of 500 sequences per virus retrieved from GenBank from different geographic regions. Annealing temperature, primer and probe concentrations were optimised, and recombinant plasmids carrying the target fragments served both as positive controls and as quantitative standards. The assay was specific for AIV and NDV, with no cross-reactivity with infectious bronchitis virus and no signal crossover between channels; its analytical sensitivity reached approximately 10-20 RNA copies per reaction. Qualitative results on reference isolates were fully concordant with two commercial veterinary kits (Cohen's κ = 1.00), and the assay passed inter-laboratory commission testing. This study establishes the design and core analytical performance of a laboratory-developed duplex assay; expanded in silico inclusivity on contemporary sequences, a broader specificity panel, inhibitor-containing matrices, an internal amplification control, and prospective clinical validation are required before routine surveillance deployment.