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 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.
Lo
Constance L H
CL
Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, China.
Yip
Shea Ping
SP
Cheng
Peter K C
PK
To
Tony S S
TS
Lim
Wilina W L
WW
Leung
Polly H M
PH
D001483:Base Sequence; D003116:Color; D017931:DNA Primers; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D044366:Transition Temperature; D010948:Viral Plaque Assay; D014773:Virology / Q000379:methods
D017931:DNA Primers; D012367:RNA, Viral