Rapid detection and typing of Dengue virus by single-tube nested multiplex-PCR / 第二军医大学学报

ABSTRACT

Objective: To establish a single-tube nested multiplex-PCR assay for rapid detection and typing of Dengue viruses for multiple infections with different serotypes. Methods: A 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. Results: By 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. Conclusion: Single-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.