Detection of Dengue Viral RNA in Mosquito Vectors by Mixed Phase and Solution Hybridization

A mixed phase hybridization technique was developed to detect dengue virus type 2 (DEN-2) RNA in pools of infected Aedes albopictus mosquitoes using radiolabelled RNA probes. This technique used a guanidine thiocyanate extraction procedure to simplify analyte preparation. The probes contained sequences complementary to portions of the NS-1 or NS-5 genes of the DEN-2 viral genome. One infected mosquito in a pool of 25 could be detected in approximately 48 h. RNAs from DEN serotypes 1-4 were extracted from cultured mosquito (C6/36) cells. The NS-1 RNA probe was highly specific for DEN-2 RNA. The NS-5 RNA probe detected both DEN-2 and DEN-4 RNA. DEN-2 RNA was also detected by molecular hybridization in concentrated solutions of guanidine thiocyanate using the NS-1 probe. Solution hybridization was 10-fold more sensitive when detecting RNA from purified DEN-2 virus than the mixed phase assay and could detect one infected mosquito in a pool of 25 within 6-8 h. Solution hybridizations were performed in 2-3 h vs 16-20 h for mixed phase hybridizations, and solution hybridizations required 5-10 times less mosquito RNA than mixed phase hybridizations to attain comparable sensitivities. However, solution hybridizations did result in a broader probe specificity than mixed phase hybridizations.