# High-sensitivity multiplex real-time RT-PCR for universal detection and virulence typing of Newcastle disease virus in poultry field samples

## TL;DR

A new real-time RT-PCR test can detect all types of Newcastle disease virus and distinguish between harmful and harmless strains in poultry.

## Contribution

A one-step multiplex real-time RT-PCR assay for universal detection and virulence typing of Class II NDV in a single reaction.

## Key findings

- The assay detected as few as 10-100 copies of NDV per tube with no cross-reactivity to other avian viruses.
- It achieved 100% concordance with established molecular assays when tested on 97 field isolates and seven reference strains.
- The assay captured NDV lineages with up to 23.7% divergence in HN genes and 13.4% in F genes.

## Abstract

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 101.0 EID₅₀ /0.1 mL for HN target and 102.0EID₅₀/0.1 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.