Quantitative RT-PCR for DENV-2 Viral Load

The RT–qPCR was performed in a 25 μl volume containing 6.25 μl of 4X TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific), 0.5 μl of 20 μM forward and reverse primers, 0.5 μl of 20 μM probes, 5 μl of viral RNA being tested, and 12.5 μl RNAse-free water using the QuantStudioTM 7 Flex System (Applied Biosciences). The primers (D2-1929, cD2-2116) and probe (D2-2000VIC-MBG/NFQ quencher) anneal at envelope (E) gene of the DENV-2 and their sequences have been reported previously [10 (link), 14 (link)]. The viral copy number was determined using a standard curve generated by an in-vitro transcribed DENV-2 RNA as previously described [10 (link)]. The concentration of standard RNA was determined based on the Certificate of Analysis received from Pharmaceutical Product Development, a global contract research organization. Briefly, a high concentration stock of DENV-2 RNA transcript obtained from CDC was tested by droplet digital PCR and was further diluted and quantified by RT–qPCR against standards previously measured by Ribogreen assay (Thermo Fisher Scientific) to make stocks at 2.7 × 10⁷ copies/μl. The stock was 10-fold serially diluted to generate the standard curve in the RT–qPCR to measure the concentration of the samples.