Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay

Abstract

A rapid and specific test was developed for the diagnosis of peste des petits ruminants disease. This assay is based on the rapid purification of RNA on glass beads followed by the reverse transcription-polymerase chain reaction (RT-PCR). To that effect, a set of primers (NP3/NP4) was used to amplify specifically a fragment of about 350 bp in the 3' end of the RNA messenger that encodes the nucleocapsid protein of the peste des petits ruminants virus. The PCR-product was detected by UV illumination after electrophoresis on agarose gel or by hybridisation with a digoxigenin-11-dUTP labelled oligonucleotide probe after a blot transfer. In comparison with the conventional titration technique on Vero cells, this RT-PCR assay was 1000-fold more sensitive. Compared with the popular Chomczynski and Sacchi's method [Anal. Biochem. 162 (1987) 156], the purification of the RNA on the glass beads offers the advantage of being more rapid and also avoiding the use of solvents.