Abi prism 7500 fast thermocycler

Blood was collected from the caudal vein of anaesthetized fish and placed in BD Microtainer^® SST™ tubes, allowed to clot for 30 min at room temperature, and centrifuged (8000× g, 2 min). Sera were collected, aliquoted and stored at −80 °C until use. RNA from serum samples was extracted using an RNeasy^® 96 Kit (Qiagen, Oslo, Norway) according to the manufacturer’s instructions (elution volume: 50 μL per RNA sample). RT-qPCR analyses were performed with a Verso™ 1-step RT-qPCR Low-ROX Kit (Thermo Scientific, Oslo, Norway) using 4 μL of RNA per sample. Primer and probe concentrations for nsP1 assay [20 (link)] were: Fwd. primer: 900 nM, Reverse primer: 900 nM, Probe: 260 nM. Reactions were run in an ABI PRISM^® 7500 FAST Thermocycler from Applied Biosystems (AB) at the following conditions: 50 °C for 30 min; 95 °C for 15 min; 40 cycles of: 95 °C for 15 s, and 60 °C for 60 s.

ZIKV was detected by RT-qPCR (TaqMan), as described by Faye et al. (2013) [17 (link)]. Two forward and reverse consensus primers (Zika_qRT_F and Zika_qRT_R), complementary to the genes coding for the NS5 protein were used at a concentration of 200 nM, along with the probe (Zika_qRT_P) at a concentration of 125 nM (S1 Table).
RT-qPCR reactions were performed for each sample as follows: 2.5 μL of TaqMan FAST 1-Step Master Mix (2x) (Applied Biosystems, Foster City, USA), 0.2 μL of 200 nM Zika_qRT_F primer (Invitrogen, USA), 0.2 μL of 200 nM Zika_qRT_R primer (Invitrogen, USA), 0.2 μL of 125 nM Zika_qRT_P probe (Applied Biosystems, Foster City, USA), 1.9 μL of nuclease-free water (Promega, Madison, USA), and 5 μL of the extracted RNA were mixed and added to each well of a 96-well optical PCR plate (Applied Biosystems, Foster City, USA). The reactions were performed using an ABI Prism 7500 Fast thermocycler (Applied Biosystems, Foster City, USA). The PCR program consisted of an initial cycle at 50°C for 5 min for reverse transcriptase activation, followed by enzyme inactivation and initial denaturation at 95°C for 20 seconds, 40 cycles at 95°C for 15 seconds for denaturation, primer annealing at 60°C for 1 min, extension at 60°C for 1 min, and final extension at 60°C for 1 min.