Differentiation of virulent and non-virulent strains of Newcastle disease virus within 24 hours by polymerase chain reaction

TLDR: It is concluded that the RT-PCR described can be used to confirm diagnosis of Newcastle disease within 24 h using RNA isolated directly from tissue homogenate by using the reverse transcrip-tase-polymerase chain reaction (RT- PCR).

Abstract: Fast diagnosis of Newcastle disease is a prerequisite for confining outbreaks. Diagnosis implies the differentation of virulent and non-virulent Newcastle disease viruses (NDV). However, conventional methods, i.e. isolation of the virus and determination of the intracerebal pathogenicity index, take at least 5 days. Therefore, we investigated whether diagnosis can be performed by using the reverse transcrip-tase-polymerase chain reaction (RT-PCR) on RNA isolated directly from tissue homogenate. Two oligonucleotide primers, representing the sequence at the cleavage site of the F protein of either virulent or non-virulent NDV strains, respectively, were used to differentiate NDV. Using the RT-PCR we were able to differentiate 15 NDV reference strains, 11 of which were virulent and 14 non-virulent. The RT-PCR was further validated by using homogenate of brain, trachea, lung and spleen from 12 chicken flocks and one turkey flock suspected of Newcastle disease. The RT-PCR detected virulent NDV in samples of seven flocks and non-virulent NDV in two out of three flocks in agreement with conventional methods. However the RT-PCR failed to detect virus in 1/3 flocks from which non-virulent virus was isolated. The results are discussed. We conclude that the RT-PCR described can be used to confirm diagnosis of Newcastle disease within 24 h using RNA isolated directly from tissue homogenate.