Anchored Pan Dengue RT‐PCR and Fast Sanger sequencing for detection of Dengue RNA in human serum

Abstract A large number of human infections are caused by different dengue virus strains, mainly in the tropical and subtropical parts of the world, but also outside the endemic regions. RT‐PCR methods are used widely for detection of dengue virus RNA in acute‐phase serum samples; however, new sequence variation can inhibit these methods. An assay was developed integrating an anchored Pan Dengue RT‐PCR with a new Fast Sanger sequencing protocol. For broad detection and identification of dengue virus RNA, including new strains of all serotypes, the conserved 3′ genome end was targeted for highly specific cDNA synthesis. A combination of degenerated primers was used for second strand synthesis, followed by tag primed amplification. The mixture of generated amplicons was identified directly by the Fast Sanger sequencing from the anchored 3′ genome end. Evaluating the assay on human serum RNA spiked with viral RNA representing the four dengue serotypes demonstrated a detection limit of 44–124 copies viral RNA per reaction for a two‐step format of the anchored Pan Dengue RT‐PCR and 100–500 copies for a one‐step protocol, respectively. The different serotypes were clearly identified from the generated sequences. Further, the 5‐hr procedure was evaluated and compared to standard real‐time RT‐PCR protocols on acute‐phase serum samples from patients with confirmed dengue infections. This assay demonstrates a strategy for virus detection, which combines nucleic acid amplification adapted for dengue virus RNA with direct and rapid sequencing. It provides a tolerance for new sequence variation and the strategy should be applicable for other RNA viruses. J. Med. Virol. 82:1701–1710, 2010. © 2010 Wiley‐Liss, Inc.