Aim: Newcastle
disease is still one of the major threats for poultry industry allover the
world. Therefore, attempt was made in this study to use the SYBR Green I
real-time PCR with melting curves analysis as for detection and differentiation
of NDV strains in suspected infected birds.
Materials and Methods: Two
sets of primers were used to amplify matrix and fusion genes in samples
collected from suspectly infected birds (chickens and pigeons). Melting curve
analysis in conjunction with real time PCR was conducted for identifying
different pathotypes of the isolated NDVs. Clinical samples were propagated on
specific pathogen free ECE and tested for MDT and ICIP.
Results: The
velogenic NDVs isolated from chickens and pigeons were distinguished with mean
Tm 85.03±0.341 and 83.78±0.237 respectively for M-gene amplification and for
F-gene amplification the mean Tm were 84.04±0.037 and 84.53±0.223. On the other
hand the lentogenic NDV isolates including the vaccinal strains (HB1 and LaSota)
have a higher mean Tm (86.99±0.021 for M-gene amplification and 86.50±0.063 for
F-gene amplification). The test showed no reaction with unrelated RNA samples.
In addition, the results were in good agreement with both virus isolation and
biological pathotyping (MDT and ICIP). The assay offers an attractive
alternative method for the diagnosis of NDV that can be easily applied in
laboratory diagnosis as a screening test for the detection and differentiation
of NDV infections.
Conclusion: As was shown by the successful rapid
detection and pathotyping of 15 NDV strains in clinical samples representing
velogenic and lentogenic NDV strains, and the agreement with the results of
virus isolation , biological pathotyping and pathogenicity indices. The results
of this report suggests that the described SybrGreen I real-time RT-PCR assay in
conjunction with Melting curve analysis used as a rapid, specific and simple
diagnostic tools for detection and pathotyping of different NDVs in clinically
infected birds. Keywords: lentogenic, melting temprature, Newcastle
virus, syer green I, velogenic
