Development of real-time reverse-transcription PCR for detection of mesogenic and virulent Newcastle disease virus RNA

According to the sequences of fusion protein (F) gene that code for the cleavage site, oligonucleotide primers and probe were designed and a real-time reverse-transcription PCR (RRT-PCR) was developed to diagnose Newcastle disease(ND) and identify mesogenic virulent NDV isolates. The positive results were obtained when the RRT-PCR assay used to detect the samples containing mesogenic virulent NDV isolates, including genotypeⅠ、Ⅵ、Ⅶ、Ⅸ. The negative results were obtained from common vaccine strains used in commercial poultry in China, including La Sota, Clone 30,B1 and V4.Similarly, negative results were obtained from samples containing infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus and RNA extracted from normal chicken tissues. F gene probe sets were capable of detecting approximately 60 genome copies with in vitro-transcribed RNA. The sensibility of RRT-PCR for detection NDV is 10 folder high than RT-PCR which we previously developed. The RRT-PCR test was used to examine clinical samples from poultry naturally infected with NDV. Overall, a positive correlation was obtained between the RRT-PCR results and virus isolation for NDV from clinical samples.