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

Abstract

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype- and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60 degrees C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 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 serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

FIG. 1

Alignment of dengue virus 3′ UTR, target area of the dengue virus group assay. Primer positions are indicated in blue, and 6-FAM- and JOE-labeled probe positions are indicated in black and green, respectively.

FIG. 2

(A) Dengue virus group-specific assay. Den-1 to -4, JEV, and YF are shown. Results represent typical amplification plots obtained with the dengue virus group-specific assay. The PE 7700 instrument reading, set to FAM filter, shows specific detection of all four serotypes but not JEV and YF. The probe with Den-2 and Den-4 specificity has a JOE fluorochrome label but is still detected with the FAM filter. (B) A log plot of Den-1 stock virus tested by the group assay. Results represent typical amplification plots obtained with the dengue virus group-specific assay. Correlation coefficient = 0.988 (FAM filter). (C) A log plot and standard curve of Den-1 stock virus tested by the group assay. Correlation coefficient = 0.988 (FAM filter). Results represent a typical standard curve graph where the threshold cycle is plotted against the starting quantity (PFU/ml).