Abstract
Dengue fever is a serious disease that threatens 40% of the world’s population, and without a vaccine or therapy, accurate diagnosis is critical in trying to save patients. Our work establishes an alternative and more specific set of oligonucleotides for use in detecting the dengue viral genome via semi-quantitative nested PCR using a relatively inexpensive enzyme and a quantitative PCR (qPCR) method using SYBR Green. To validate the DENV detection methods, assays were performed to ensure the specificity and sensitivity by generating five amplicons of standard virus samples obtained from infected Vero cell cultures. Semi-quantitative nested PCR and SYBR Green PCR techniques are important tools in the detection and quantification of the four serotypes of dengue virus and can be used for dengue virus detection in mosquito vectors, as well in serological or other human fluids, in much the same way as tests for dose-responsiveness of antivirals in laboratory cell cultures and viral load determinationsare performed in other types of studies.
Highlights
Dengue fever arthropod-borne disease is transmitted by Aedes and caused by dengue virus (DENV), a member of the Flavivirus genus and Flaviviridae family
Brazil faces 3 simultaneous epidemics caused by the Zika virus (ZIKV), Chikungunya virus (CHIKV) and DENV, in addition to some cases of sylvan yellow fever virus (YFV) infections, all of which are transmitted by the Aedes aegypti mosquito
We propose an experimental procedure using semi-quantitative nested polymerase chain reaction (PCR) using a less expensive enzyme and a quantitative PCR method using SYBR Green
Summary
Dengue fever arthropod-borne disease is transmitted by Aedes and caused by dengue virus (DENV), a member of the Flavivirus genus and Flaviviridae family. Brazil faces 3 simultaneous epidemics caused by the Zika virus (ZIKV), Chikungunya virus (CHIKV) and DENV, in addition to some cases of sylvan yellow fever virus (YFV) infections, all of which are transmitted by the Aedes aegypti mosquito. In this context, the clinical and epidemiological diagnoses of these diseases are difficult since their symptoms are very much alike [6]. There are 4 dengue serotypes (DENV1-4), which are genetically similar and share approximately 65% of their genomes This diagnostic test takes time, making medical decisions difficult.
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