Development of a qPCR system to detect dengue virus from mosquito samples

RIPOLL, LUCAS; GHIRINGHELLI, DANIEL; BERGIER, JULIAN; BILEN, MARCOS

Salta

Simposio; III Simposio Latinoamericano de Virología Ambiental; 2016

Dengue fever is an endemic disease in South America. In 2015 the Panamerican Health Organization confirmed almost 500,000 cases in southern region. In Argentina, the diagnostic methods most widely used are serological assays, cell culture in C6/36 cells, or molecular analysis by RT-PCR. The use of methodologies based on real time PCR for detection of Dengue has a limited application in health centers, due to the high costs of imported reagents. This study presents the development of a detection system based on qPCR, where primers and probes are from original design, and reaction components are produced by a local company. The primers and probes used in this work were designed from a comprehensive bioinformatic analysis, looking from 80 to 90 complete genomes sequences of each viral serotype. This information was used to generate consensus oligonucleotides and specific probe for each serotype. As a result of this work, we highlight the obtaining of an optimized system for the detection Dengue virus and each serotype. Analytic validation showed 100% specificity for each serotype and sensitivity for detection of 10-100 target molecules. The system was tested in mosquito samples, using in vitro synthesized RNA. This system would be a sensitive and powerfull tool for detect the virus in the vector and provide information about possible outbreak future.