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 10^2.0EID₅₀/0.1 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 (¹¹²RRQKRF¹¹⁷, ¹¹²RRRKRF¹¹⁷, ¹¹²KRRKRF¹¹⁷, and ¹¹²RRQRRF¹¹⁷) were positive for both targets, whereas those with monobasic motifs (¹¹²GKQGRL¹¹⁷ and ¹¹²GRQGRL¹¹⁷) 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.