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Title: 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
Authors: Lo, C
Yip, SP 
Cheng, P
To, T 
Lim, W
Leung, P 
Issue Date: 2007
Publisher: American Association for Clinical Chemistry
Source: Clinical chemistry, 2007, v. 53, no. 4, p. 594-599 How to cite?
Journal: Clinical chemistry 
Abstract: 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 (Tms).
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.
ISSN: 0009-9147
EISSN: 1530-8561
DOI: 10.1373/clinchem.2006.077446
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