Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis

In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 × 102 plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (Tms): 89.23 ± 0.27 °C for velogenic strains, 90.17 ± 0.35 °C for pigeon mesogenic strains, 91.25 ± 0.14 °C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.