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Background:Dengue virus, transmitted by mosquitoes, causes every year 50 million cases of dengue fever. A standardize method for early diagnosis is still needed for clinical diagnosis and epidemiological studies.

Objective: To develop and validate for sensitivity, specificity, linearity and precision real-time one-step RT-PCR for the detection of dengue viruses.

Study design: Multiple alignments of dengue virus sequence for each serotype were done and used to develop five systems of real-time RT-PCR to detect all dengue virus strains and then identify the serotype. These systems were validated on synthetic RNA transcripts for specificity, sensitivity, precision and linearity and then applied on series of human samples.

Results: The specificity of each system was determined by sequence alignments and experimentally tested on different flaviviruses. Methods precision and linearity were statistically validated. Each of these systems allowed the detection of less than one infectious particle and was able to detect and serotype quickly dengue virus in human samples where infectious virus cannot be isolated anymore.

Conclusions: These systems are valuable tools for dengue virus diagnosis and epidemiological studies. Standardization and validation of these methods allow an easy transfer to diagnostic laboratories.