Background: Confirmation of Dengue virus [DV] infection by laboratory and serotyping is critical information for patient's management and to applying public health strategies to control the Aedes aegyptis vector. Molecular biology methods have largely displaced the classical cell culture techniques reducing the time of reporting. The aim of this study was standardized a Real-Time PCR assay for detection, serotypification and quatification of the DV using SYBR® green I RT-PCR technologies. Methods: DV serotypes (DV1-4) were selected from the laboratory collection and human serum/blood specimens were collected from febrile patients clinically suspected of DV infection (WHO criteria), during the first three days of clinical symptoms. Total RNA were extracted from the patient samples using a QIAmp®RNA Viral/Blood Mini-Kit (QIAGEN®). Three different universal primers designed to amplified pre-membrane (CprM) and nonstructural protein 5 (NS5) were used during the detection assay. Subsequently, the viral types were determined using the CprM forward primer and a specific reverse primer for each virus serotype [DV1-DV4]. The Reverse Transcription and amplification assays were performed in one step protocol using SYBR® Green-I RT-PCR kit (QIAGEN®) in a 38 cycle process. Results: All viral types were amplified with the three universal assays. The average CT values for the three region and four-serotypes showed a CprM primer of 26,5; First NS5 region (3NC) 15,9, and finally for Second NS5 region (JMC) 20,9. When the specific reverse primers for DV1-4 were used, only the amplification occurs in the corresponding viral serotype. Validation assay was performed in two hundred-eight serum samples from febrile patients, 33.7% (70/208) of them were positive by RT-PCR. Genotyping showed DV2 (31,1%) as the most frequent genotype, followed by DV1, DV3 and DV4 and a significant difference was observed in viral load in patients with DHF and SSD. Conclusion: We compare the efficiency of the amplification of three different regions of Dengue Virus using a Universal SYBR® green I RT-PCR assay. Amplification of First NS5 region (3NC) showed the best amplification efficiency in patients and controls. Serotyping assay showed the circulation of DV1-DV4 in our communities. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive