Reverse transcriptase assay colorimetric kit

The assessment of the anti-HIV-1 RT potential of different plant extracts was done colorimetrically by using Reverse Transcriptase Assay Colorimetric Kit (Roche, USA). The experiments were performed according to the instruction given in the user manual. Briefly, the experiments were performed by adding 20 μL of HIV-1 RT solution, 20 μL plant extract, and 20 μL of reaction mixture containing a hybrid of template (poly rA) and oligo dT (primer), dTTP, Mg²⁺, and buffer. One set of reaction was carried out without plant extract. The reaction mixture was incubated at 37°C for 1 h. The whole reaction mixture was transferred into 96-well plate. This system was left for incubation at 37°C for 1 h. The solution was removed completely, and the plate was washed 5 times for 30 seconds each time with washing buffer. In the next step, 200 μL of antidigoxygenin- (DIG-) peroxidase (POD) (working solution) was added. It was again incubated at 37°C for 1 h. After incubation, the wells were washed 5 times in 30 seconds with washing buffer. In the further step, 200 μL of ABTS substrate solution was added per well and incubated at 25°C for 30 min. In the final step, OD was measured at 405 nm (reference λ = 490 nm) in an ELISA plate reader.

A luciferase-expressing EIAV reporter virus was constructed by modifying a three-plasmid EIAV transfection system (54 (link)). HEK293T cells were cotransfected with pONY8.1-LUC and pEIAV-GagPol and pcDNAenv or pcDNAenvT. The reporter virus was collected 48 hpt, centrifuged at 1,000 rpm for 10 min to remove cell debris, filtered through a 0.45-μm-pore filter unit, and stored at –80°C. The luciferase reporter virus was quantified using a reverse transcriptase (RT) activity kit (reverse transcriptase assay, colorimetric kit; Roche, Switzerland) and used to infect HEK293T/ELR1 cells (an equine lentivirus receptor 1 [ELR1]-expressing HEK293T cell line) (54 (link)), FDD cells, and eMDMs in 96-well plates for 48 h. These seeded cells were washed and subjected to luciferase analysis.