Quantitative PCR for Zika Virus Detection

Quantitative PCR (qPCR) was performed according to established methodologies [13 (link)]. Briefly, we used the QuantiTect Probe RT-PCR Kit (Qiagen) reagents and the CFX96 Real-Time System (Bio-Rad) where each 25 μL PCR reaction contained 12.5 μL 2X QuantiTect PCR mastermix, 1 μL of each 10 mM primer, 0.5 μL 0.2 mM probe, 0.5 μL reverse transcriptase, 3 μL RNA template and 6.5 μL H₂O. Every PCR was performed as follows: reverse transcription at 50°C for 30 min, initial PCR activation at 95°C for 5 min and 45 amplification cycles consisting of a 95°C denaturation for 10 sec and a 60°C annealing/extension for 30 sec. Sequences of primers and probes are as follows; ZIKV-F: 5'- TGG TCA TGA TAC TGC TGA TTG C -3', ZIKV-R; 5'- CCT TCC ACA AAG TCC CTA TTG C -3', ZIKV-probe5'- /56-FAM/CGG CAT ACA GCA TCA GGT GCA TAG GAG /3BHQ_1/ -3', Vero (African green monkey) GAPDH-F:5′- GGG TGT GAA CCA TGA GAA GTA T-3′, GAPDH-R; 5′- GAG TCC TTC CAC GAT ACC AAA G-3′ and GAPDH-probe: 5'- /5HEX/AC AAC AGC CTC AAG ATC GTC AGC A/3BHQ_1/ -3'. The relative amount of viral transcript to GAPDH was calculated using the 2^-ΔΔCT method. Data were expressed as fold change RNA compared to the control.