A Duplex Real-Time RT-PCR Assay for Profiling Inhibitors of Four Dengue Serotypes

Abstract

We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, β-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells β-actin gene is 100%.

Development of a Novel Chikungunya Virus-Like Replicon Particle for Rapid Quantification and Screening of Neutralizing Antibodies and Antivirals

“…qRT-PCR was conducted using the ABI StepOnePluses real-time PCR system (Applied Biosystems) in triplicate. The primer sequences used in this assay were as follows: Actin-F, 5′-GGC CAG GTC ATC ACC ATT-3′; Actin-R, 5′-ATG TCC ACG TCA CAC TTC ATG-3′ ( 46 ); 3′-UTR-F, 5′-CAA ACG GAA GAG ATG TAG G-3′; and 3′-UTR-R, 5′-GGG TTC GGA GAA TCG TGG-3′. Estimates of 3′-UTR expression were based on relative quantification using the comparative critical threshold (CT) method.…”

Macrolides May Prevent Severe Acute Respiratory Syndrome Coronavirus 2 Entry into Cells: A Quantitative Structure Activity Relationship Study and Experimental Validation

“…Six hours later, cell lysates were prepared using Trizol reagent (Thermo Scientific). Viral RNA content was determined by RT-qPCR using previously validated sets of primers and probes specific for the detection of the SARS-CoV-2 E gene and the cellular β-actin gene, for normalization purposes. The δCt method was used for relative quantitation of the intracellular viral RNA accumulation in compound-treated cells compared to the levels in infected cells treated with DMSO, set as 100%.…”

A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR

“…These reagents have garnered increasing interest as tools for enabling high-throughput gene-expression analysis34. Recent studies have validated the accuracy of RT-qPCR relying on commercial cell-lysis reagents56, providing justification and incentive for expanded use.…”