Received on July 29, 2006
Accepted on January 25, 2007

Molecular Diagnostics and Genetics

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

¹ Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China
² Public Health Laboratory Centre, Centre for Health Protection, Department of Health, Hong Kong SAR, China

^* To whom correspondence should be addressed. E-mail: htpolly{at}inet.polyu.edu.hk.

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_ms).

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 °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 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 T_m and color multiplexing was developed for detecting and typing all 4 DV serotypes.