Amplification of dengue 2 virus ribonucleic acid sequence using the polymerase chain reaction

The polymerase chain reaction (PCR) has been adapted to the amplification of dengue type 2 virus (DEN2) nucleic acid sequences. A pair of 20-mer oligonucleotides were designed and synthesized based on conserved sequence blocks of DEN2 strains isolated from different geographical areas. RNA samples were prepared from two DEN2 strains, prototype New Guinea C (NGC) and local isolate Hainan 98 (HN98). The reverse transcription step was performed for cDNA synthesis before the standard PCR procedures. The amplified products were fragments about 476 bp in length, corresponding to the upper one third of DEN2 envelope gene (E1 to E476 nt). Specificity of the amplification products was confirmed by "nested" PCR using the internal primers and by Southern and dot blot hybridization to cloned DEN2 cDNA probes following agarose gel electrophoresis. Further improvement and the potential application of the methods in study of dengue virus RNA are discussed.