ObjectiveTo establish a single-tube nested multiplex-PCR assay for rapid detection and typing of Dengue viruses for multiple infections with different serotypes. MethodsA pair of outer universal primers designed for all the four Dengue virus serotypes were used to amplify the mixed RNA of 1-4 dengue viral serotypes by one-step RT-PCR，and the products were used as template for nested multiplex PCR using four pairs of serotype-specific primers in the same reaction tube. The sensitivity and specificity of single-tube nested multiplex PCR assay amplifying from the mixed 1-4 serotype dengue viral RNA were subsequently compared with those amplifying from the single serotypes dengue viral RNA. ResultsBy optimizing the reaction condition，four specific fragments (482,119,290,and 389 bp) were successfully amplified from the mixed RNA of 1-4 serotypes dengue viruses in single tube by single-tube nested multiple-PCR. Its sensitivity and specificity amplifying from the mixed RNA of 1-4 serotypes dengue viruses were similar to those amplifying from the single serotype dengue viral RNA. The detection limit of nested multiple-PCR was 66.068 copies/μl. ConclusionSingle-tube nested multiple-PCR method is simple,rapid,sensitive, and specific for detecting and typing dengue viruses,and it is valuable for detecting and typing of the clinical multiple infections.