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 detecting 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(m)s).
Results: Fluorescence signals with specific emission wavelengths were obtained from DEN-1 and DEN-3. SGI melting profiles showed a T(m) difference between DEN-2 and DEN-4 of 4.7 degrees 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 x 10(-4), 1.05 x 10(-3), 8.15 x 10(-4), and 5.80 x 10(-3) plaque-forming units per reaction, respectively. The assay had a dynamic range of 10(3)-10(8) plaque-forming units/L and could be performed in 2 h.
Conclusions: A single-tube, 1-step reverse transcription-PCR assay based on T(m) and color multiplexing was developed for detecting and typing all 4 DV serotypes.