Detection of Newcastle disease virus RNA in infected allantoic fluids by in vitro enzymatic amplification (PCR)

Abstract

The Polymerase Chain Reaction (PCR) procedure was applied in order to identify the Newcastle disease virus (NDV), an avian paramyxovirus (A-PMV 1). The sequence selected for amplification consists of 238 bp lying in the gene encoding the fusion protein F. A pair of 19-mer and 18-mer oligonucleotides, flanking this sequence, were used as primers. Following RNA extraction by the proteinase K method, a cDNA was prepared using the previous 19-mer oligonucleotide as the primer. The amplification reaction product was analyzed by electrophoresis and ethidium bromide staining, using the restriction enzymes HaeIII, Mbo II, and Nar I. The PCR was performed on cDNA prepared from 30 A-PMV 1 and 3 other strains (A-PMV2, A-PMV3, A-PMV4). It was thereby demonstrated that the selected sequence was highly specific and constant. However, two of the PMV1 strains isolated from feral ducks, are thought to present a deletion of about 25 bp inside this fragment as shown by the smaller length of the corresponding amplified product and the disappearance of the NarI restriction site. The advantages of this technique, as a first step in evaluating virulence by means of molecular biology, is discussed.