Detection of dengue virus RNA using nucleic acid hybridization

Abstract

Conditions for using slot-blot nucleic acid hybridization to quantitatively detect dengue-2 virus using a radiolabelled cDNA probe, pVV17, were determined. As little as 11 plaque-forming units of virus were detected using a hybridization mixture without formamide and performing the test at 70 degrees C. While predominantly serotype-specific using stringent (65 degrees C) washing conditions, the probe detected all four dengue virus serotypes using astringent (28 degrees C) washing conditions. No significant qualitative differences were detected using Thai dengue-2 viruses isolated over a 10-year period. High titered, anti-flavivirus antibodies blocked virus detection by an antigen capture, enzyme-linked, immunosorbent assay or by intrathoracic inoculation of Toxorhyncites mosquitoes, but not by nucleic acid hybridization. The appearance of virus-specified RNA coincided with the detection of antigen in infected C6/36 (Aedes albopictus) cells by immunofluorescence, or in cell culture supernatants by the antigen capture method. The method has potential as a diagnostic tool for identifying dengue viruses in clinical and field specimens.