One-step rapid reverse transcription-PCR assay for detecting and typing dengue viruses with GC tail and induced fluorescence resonance energy transfer techniques for melting temperature and color multiplexing

Background: Dengue fever is an arthropod-borne infection caused by dengue viruses (DVs; DEN-1 to DEN-4). Early diagnosis is critical to prevent severe disease progression and the spreading of DV because no vaccine or specific treatment is available; therefore, a rapid and specific diagnostic assay capable of defecting and typing all serotypes would be ideal. Methods: We amplified RNA samples from all 4 DV serotypes and Japanese encephalitis virus with 4 serotype-specific forward primers and a universal species-specific reverse primer. DEN-1 and DEN-3 forward primers were labeled at their 5′ ends with BODIPY 630/650 and Cy5.5, respectively. DEN-1 and DEN-3 amplicons were detected by their characteristic emission generated from induced fluorescence resonance energy transfer. The presence of DEN-2 and DEN-4 amplicons was indicated by SYBR Green I (SGI) signals at specific amplicon melting temperatures (T ms). Results: Fluorescence signals with specific emission wavelengths were obtained from DEN-1 and DEN-3. SGI melting profiles showed a Tm difference between DEN-2 and DEN-4 of 4.7°C, which was sufficient for differentiating these 2 serotypes. The primers did not amplify the Japanese encephalitis virus. The detection limits of DEN-1 to DEN-4 were 1.64 × 10-4,1.05 × 10-3, 8.15 × 10-4, and 5.80 × 10-3 plaque-forming units per reaction, respectively. The assay had a dynamic range of 103-108 plaque-forming units/L and could be performed in 2 h. Conclusions: A single-tube, 1-step reverse transcription-PCR assay based on Tm and color multiplexing was developed for detecting and typing all 4 DV serotypes.