Optimization and Validation of Universal Real-Time RT-PCR Assay to Detect Virulent Newcastle Disease Viruses

Newcastle disease, caused by virulent strains of avian paramyxovirus 1 (APMV-1), occurs globally and has significant social and economic impact. APMV-1 is a rapidly evolving RNA virus and is genetically divided into class I and class II with almost all virulent viruses being of class II. The considerable genetic diversity of the virus adds complexity to maintaining the high sensitivity and specificity of molecular detection assays. The current USDA's fusion gene rRT-PCR assay was designed for class II APMV-1 isolates with an emphasis on early-2000s US strains. Assessment with globally circulating genotypes confirmed previously described lower sensitivity (sub-genotypes VII.1.1, VII.2) and identified absence of detection (genotype XIV). An additional forward primer and two probes were designed using a comprehensive complete fusion gene sequence database. The optimized multiplex assay detected genotype XIV and improved sensitivity for sub-genotypes VII.1.1 and VII.2, with maintained sensitivity for the remaining genotypes. No near-neighbors or APMV-1 of low virulence were detected. Using field and experimental clinical samples, both the specificity and sensitivity were determined to be 100%, compared to the current assay with 100% and 93%, respectively. The new assay identifies all known chicken virulent APMV-1 genotypes with the benefit of using an exogenous internal positive control, which monitors extraction efficiency and inhibitors.