Detection of flavivirus RNA in infected cells using photobiotin-labelled hybridization probes

Ten plasmids containing viral cDNA inserts of portions of the dengue virus type 2 (DEN-2) or Kunjin virus (KUN) genomes were biotinylated using photobiotin acetate and used as probes for the detection of flavivirus RNA in infected Vero cells. The viral cDNA inserts ranged in length from 0.19 to 2.7 kilobase pairs, and represented segments of the flavivirus genome coding for structural and non-structural proteins. In spot hybridization assays (hybridization at 60°C) with RNA extracted from cells infected with one of fourteen different flaviviruses or Semliki Forest virus, all DEN-2 and KUN probes hybridized specifically with RNA from cells infected with DEN-2 or KUN, respectively. At the reduced stringency of lower temperatures, specific hybridization to homologous viral RNA was still a feature of the probes, and only limited cross-hybridization to the RNA of some other flavivirus species was detected.