Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PC R) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and f luorogenic probes were designed against conserved regions of the dengue vir us genome. The RT-PCR assay is a rapid single-tube method consisting of a 3 0-min RT step linked to a 45-cycle PCR at 95 and 60 degreesC that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue v iremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan ass ays were compared to virus isolation using C6/36 cells followed by an immun ofluorescence assay using serotype-specific monoclonal antibodies. Viral ti ters in sera were determined by plaque assay in Vero cells. The serotype-sp ecific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positiv e samples, for a sensitivity of 92.5%, while the group-specific assay detec ted 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the ser otype concordance of all assays compared to cell culture isolation and nega tive results obtained when 21 normal human sera and plasma samples were tes ted. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping too ls for epidemiological studies of dengue virus infections.