Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus

Abstract

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10(7) PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

FIG. 1.

Standard curves of four dengue virus serotypes tested by one-step SYBR Green-based quantitative RT-PCR using group-specific primer pair. Standard curves were generated from the amplification plots of each of the four dengue strains representing DEN-1 (A), DEN-2 (B), DEN-3 (C), and DEN-4 (D). The starting viral titer was plotted against the Ct value of each dilution.

FIG. 2.

Standard curves of four dengue virus serotypes tested by one-step SYBR Green-based quantitative RT-PCR using serotype-specific primer pair. Standard curves were generated from the amplification plots of each of the four dengue strains representing DEN-1 (A), DEN-2 (B), DEN-3 (C), and DEN-4 (D). The starting viral titer was plotted against the Ct value of each dilution.

FIG. 3.

Analysis of amplification products of SYBR Green-based real-time RT-PCR using group- and serotype-specific primer pairs. The RT-PCR products were observed in ethidium bromide-stained agarose gel. Input viral RNAs were extracted from each of the 16 representative dengue virus strains as shown in Table 2. Results from each of the DEN-1 (A), DEN-2 (C), DEN-3, and DEN-4 (D) serotypes are shown. The 171-bp amplicons appearing in all panels were the expected products for all four dengue serotypes using a group-specific primer pair (DN-F-DN-R), whereas the 193-, 204-, 204-, and 132-bp amplicons were the expected products for each of the DEN-1, DEN-2, DEN-3, and DEN-4 serotypes, respectively. N, nontemplate control.

FIG. 4.

Comparisons of dengue viral titers between the serum samples negative for dengue virus by virus isolation and positive for dengue virus by the SYBR Green-based RT-PCR method (VI-PCR+) and those positive by both virus isolation and the SYBR Green-based RT-PCR method (VI+PCR+). The viral titers in the VI-PCR+ or VI+PCR+ serum samples from each of the DEN-1-, DEN-2-, DEN-3-, and DEN-4-infected patients were calculated according to the standard curves shown in Fig. 1 using group-specific primer pairs.