We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer Dl. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Dengue (DEN) viruses belong to the family Flaviviridae, genus Flavivirus, and occur as four antigenically related, but distinct serotypes designated DEN-1, 2, 3 and 4 (EG Westaway et al. 1985 Intervirology 24: 183-192). The viruses are characterized by a single strand of RNA associated with a core protein, in a nucleocapside surrounded by a lipid envelope. The genoma consist of a single open reading frame coding for core protein (C), precursor of the membrane protein (prM) and envelope (E) structural proteins, followed by the non structural proteins NS1, NS2a, NS2b, NS3, NS4a The genetic variation among DEN viruses has been demonstrated by numerous methods including oligonucleotides fingerprinting, restriction enzymes, primer extension sequencing and nucleotide sequences from different fragments of genoma (R

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