The following DENV strains from ATCC and BEI Resources, representing DENV serotypes 1-4, were quantified using the standard curves generated from the DENV 1-4 synthetic molecular standards, respectively:
DENV 1 = TH-S-man (ATCC® VR-1586™)
DENV 2 = New Guinea C (NR-84, BEI Resources)
DENV 3 = Philippines/H87/1956 (NR-80, BEI Resources)
DENV 4 = H241 (ATCC® VR-1257™)
Viral RNA was extracted using the QIAamp® Viral RNA Mini Kit (QIAGEN®), and subsequently diluted 1:100, 1:1000, and 1:10,000 for quantification via qRT-PCR. Uninfected Vero (ATCC® CCL-81™) and LLC-MK2 Derivative (ATCC® CCL-7.1™) cell lines were used as negative controls. The titers of the native DENV 1-4 samples were determined using the respective standard curves generated by the synthetic molecular standards; the primers employed were those designed for the DENV 1-4 Real-Time RT-PCR Assay developed by the CDC (Figure 2A, DENV 1-3 data not shown; Figure 2B).
Figure 2. Quantification of native DENV RNA. (A) qRT-PCR amplification plot showing the DENV-4 synthetic molecular standard (blue), the native dengue virus sample (pink), and the negative control sample (green). (B) Titers of DENV-1, -2, -3, and -4 samples as determined by the qRT-PCR standard curves generated using the DENV 1-4 molecular standards, respectively.