Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.
Total time
~4–6 hours (standard RNA synthesis + RT-qPCR runs)
Model organism
HEK293 cells (dengue virus-infected culture)
Steps
1
Synthesize DENV 3'UTR RNA standard
Generate in vitro RNA transcripts from dengue virus 3' untranslated region template to serve as quantification standards for RT-qPCR assays.
▶ 01:06
2
Prepare virus samples for RT-qPCR
Process infected cell culture supernatant directly without RNA extraction, diluting samples as needed for quantitative analysis.
▶ 03:15
3
Set up and run real-time PCR
Configure RT-qPCR reaction mixture with primers and probe, then execute thermal cycling protocol to measure viral RNA amplification in real time.
▶ 04:19
4
Analyze RT-qPCR quantification results
Generate standard curve from known RNA concentrations and determine unknown virus sample titers from cycle threshold values and standard curve regression.
▶ 06:01