Serotyping of Dengue Virus

A reverse transcriptase-polymerase chain reaction (RT-PCR) and a single-tube multiplex PCR assay was modified for typing of dengue virus in different geographical areas of Thailand during 2000-2001. A set of primers (D1 and D2) was used to generate the RTPCR product of 511 bp in size which subsequently underwent a single-tube multiplex PCR amplification using the highly specific primers for each of the dengue virus serotypes (D1, TS1, TS2, TS3 and DEN4). The PCR products of 482, 119, 290 and 392 bp in size were generated for dengue virus serotypes 1, 2, 3, and 4, respectively. Each set of specific primers showed no amplification of non-specific and non-target PCR products from human genomic DNA. The method was applied for investigation of 637 human blood samples in Thailand during 2000-2001 and found that 71, 43, 28, and 43 patients were classified as having a single infection with serotypes 1, 2, 3, and 4, respectively. Multiple infections with two or more dengue virus serotypes were also detected. and inappropriate for serotyping due to extensive cross-reactivity of antibodies among dengue viruses. The PCR was recently developed for the diagnosis of each serotypes of dengue virus (Mason et al, 1987; Deubel et al, 1990; Eldadah et al, 1991; Henchal et al, 1991; Harris et al, 1993; Yenchitsomanus et al, 1996). However, it needed a single PCR amplification for each serotype which was time-consuming and labor-intensive when a large number of samples were investigated. A clear need exists for an assay that can be performed rapidly and that is sufficiently sensitive and specific to be clinically and epidemiologically useful. Here, we have modified a simple, sensitive, and rapid method for the detection, identification and typing of dengue viruses by using a combination of a RT-PCR and a single-tube multiplex PCR that could be implemented for epidemiological studies and detection of the viruses in carriers. MATERIALS AND METHODS