Development of a novel real-time PCR-based strategy for simple and rapid molecular pathotyping of Newcastle disease virus.

A novel real-time PCR strategy was applied to simultaneously detect and to discriminate low-pathogenic lentogenic and virulent meso/velogenic Newcastle disease virus (NDV). The pathotyping is achieved by a three-step semi-nested PCR. A pre-amplification of the cleavage site (CS) region of the F gene is followed by a two-level duplex real-time PCR directly targeting the CS, combining detection and pathotyping in a single tube. A wide range of NDV isolates spanning all genotypes were successfully detected and pathotyped. Clinical samples from outbreaks in Sweden in 2010 that were positive by the novel PCR method were also successfully pathotyped. The method is time-saving, reduces labour and costs and provides opportunities for rapid diagnosis at remote locations and in the field. Since the same strategy was also recently applied to avian influenza virus pathotyping, it shows promise of finding broad utility in diagnostics of infectious diseases caused by different RNA viruses in various hosts.