4.5. HIV-1 RT Assay

An HIV RT colorimetric assay kit (Roche Diagnostics, Mannheim, Germany) and HIV-1 recombinant RT (Merck) were used to test for the effect of O. labiatum extract on HIV-1 reverse transcription. The assay was performed according to the manufacturer’s instructions. The enzyme was incubated for 1 h with three different concentrations of the extract (25, 50 and 100 μg/mL). Subsequent 1 h incubation steps included the binding of biotin-labelled DNA to the surface of microplate modules that have been pre-coated with streptavidin, and the addition of an antibody conjugated to peroxidase that binds to the digoxigenin-labeled DNA. In the final step, the peroxidase substrate solution (2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) was added and the peroxidase enzyme catalyzed the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples was read at 405 nm with a reference wavelength of 492 nm using a microtiter plate reader (Multiskan Ascent; Thermo Labsystems) and was directly correlated to the level of RT activity in the sample. Doxorubicin (Sigma) [29], a known HIV-1 RT inhibitor, was used as a positive control. The experiment was performed for at least four independent biological repeats in triplicates.