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

Abstract

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. 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). 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. 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.