Detection and serotyping of Dengue viruses by real time RT- PCR and High Resolution Melting Analysis

QUINTANA SILVINA; ESTEVEZ SUSANA

Simposio; III International Clinical Virology Simposium and Advances in Vaccines; 2010

Dengue (DEN) fever and dengue hemorrhagic fever/dengue shock syndrome are important human arboviral diseases caused by dengue viruses. There are four serotypes: DEN-1, DEN-2, DEN-3 and DEN-4. Early diagnosis is critical to prevent severe disease progression and the spreading of DEN Virus 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. The current molecular methods available for identifying the different DEN members can be both expensive and technically demanding. The aim of this work was to develop a Real Time RT-PCR and high-resolution melting approach to detect and differentiate DEN serotypes. To this end, RNA from reference serotypes provided by "Dr. Julio I. Maiztegui" Institute was used to optimize the technique. We developed a Real Time RT-PCR assay using EvaGreen as reporter dye for the detection of viral RNA of all DEN virus serotypes. The generic primer set used in this work targeted the 3? noncoding region of DEN viruses. After the amplification, a High Resolution Melting profile analysis was done. The melting profiles showed a Tm (Melting temperature) difference between DEN-2 and DEN-3 of 2.5 °C (85, 5 and 88°C), which was sufficient for differentiating these 2 serotypes. The Tm of DEN-4 and DEN-1 was 87 °C, but the shapes of the melting curves were different. Thus, this real-time RT-PCR assay based on High Resolution Melting analysis can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid (< 4h) identification and serotyping of DEN virus isolates.