Quantification of FMDV nucleic acid in pleconaril and ribavirin treated timed sample by SYBR green based quantitative-PCR (Q-PCR)

The 24 and 48 h old timed samples treated either with pleconaril or ribavirin and were found negative in PCR, were further subjected to SYBR green based real time PCR for the detection and quantification of FMDV RNA [36]. Real time-PCR was performed using a two-step RT-PCR kit [Maxima SYBR Green/ROX qPCR Master Mix (2x), Cat # K0221, Thermo Scientific; USA] in 7500 ABI real time system (M/s Applied Biosystems). RNA extraction and cDNA synthesis were carried out as described earlier, from a FMD virus infected untreated and drug treated timed samples at different intervals. The real time PCR reaction was set up in a total volume of 20 μL using 10 μL of SYBR Green PCR Master mix, 2 μL of cDNA template, 0.6 μL (10 pmol/μL) each of gene specific forward (DHP-13) and reverse primers (NK-61) and volume was adjusted up to 20 μL with nuclease free water. Real-time PCR was done with initial denaturation at 95 °C for 10 min followed by 40 cycles of amplification with denaturation at 95 °C for 15 s., annealing at 60 °C and extension at 72 °C for 30 s. Specificity of the amplified product was assessed by dissociation curve generated at temperature 55 °C through 95 °C with 1 °C increment. The standard dilution series of FMDV O was used to construct standard curves from which the quantity of viral RNA (as viral RNA TCID₅₀ equivalent) of each timed sample treated with pleconaril, ribavirin and untreated virus control cell culture supernatants were estimated. The threshold was adjusted manually by inspecting the amplification plot and Ct values were calculated as the cycle at which the amplification curve crosses the threshold line.