High-Sensitivity Multiplex Real-Time RT-PCR for Universal Detection and Virulence Typing of Newcastle Disease Virus in Poultry Field Samples

Newcastle disease virus (NDV) remains a major global threat to poultry production despite widespread vaccination, driven by the emergence of genetically diverse and virulent Class II strains. We developed and validated a one-step multiplex real-time RT-PCR assay enabling simultaneous universal detection of all Class II NDV genotypes and differentiation between virulent (velogenic/mesogenic) and non-virulent (lentogenic/asymptomatic) pathotypes in a single reaction. The assay showed high analytical sensitivity, detecting as few as 10 1.0 EID₅₀ /0.1 mL for HN target and 10 2.0 EID₅₀/0.1 mL for F target, with limits of 10 and 100 copies/tube using plasmid standards, and exhibited no cross-reactivity with non-target avian viruses. When evaluated against 97 field isolates and seven reference strains collected over a 75-year period (1946-2021) from multiple continents, the assay achieved 100% concordance with four established molecular assays. All 80 isolates with multi-basic F protein cleavage site motifs ( 112 RRQKRF 117 , 112 RRRKRF 117 , 112 KRRKRF 117 , and 112 RRQRRF 117 ) were positive for both targets, whereas those with monobasic motifs ( 112 GKQGRL 117 and 112 GRQGRL 117 ) were detected only by the universal HN assay, consistent with a non-virulent phenotype. The assay detected NDV lineages spanning >75 years of virus evolution, from historical strains (Herts 33/56, Kr-KJW/49) to recent isolates (UPM/1051/2018, UPM111), capturing up to 23.7% nucleotide divergence in complete HN genes and 13.4% in F genes. This platform integrates broad-spectrum detection with precise virulence differentiation, offering a rapid, sensitive, and reliable tool for Class II NDV surveillance, outbreak response, and vaccine strain monitoring.