Detection of Newcastle disease virus in poultry vaccines using the polymerase chain reaction and direct sequencing of amplified cDNA

Abstract

In order to develop an in vitro method for the control of poultry vaccines for identity and the absence of extraneous agents, reverse transcription-polymerase chain reaction (RT-PCR) was applied for the detection of Newcastle disease virus (NDV). NDV vaccines were employed for the establishment of the method, using two primer pairs spanning the cleavage site of the F0 fusion protein coding sequence. Amplification of a specific cDNA segment was possible from live and inactivated, oil-adjuvanted NDV vaccines without prior treatment. The cDNA was characterized by restriction endonuclease digestion as well as by direct nucleotide sequencing. The RT-PCR was able to detect between 5 × 10 2 EID 50 (in live vaccine preparations) and 10 5 EID 50 or 0.056 haemagglutinating units of NDV (in inactivated vaccine preparations). In addition, live vaccine preparations were inactivated with β-propiolactone (β-PL). Amplified cDNA was obtained after treatment with 0.1% β-PL, whereas at a concentration of 1% or 10% no specific bands were visible in the agarose gel. These results demonstrate the applicability of the method for the control of poultry vaccines for identity and for the absence of extraneous agents, and additionally allow a rapid characterization of the respective NDV strain.