Itaq universal probes one step kit

Viral RNA was extracted using QIAmp Viral RNA Mini kits (QIAGEN) according to the manufacturer’s instructions. RT-qPCR was performed using iTaq^TM Universal Probes One-Step kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s guidelines. ARPV primers ARPV-7562F (5′-CGGTGTTCATTGAGGATGAC-3′), ARPV-7714R (5′-TGATACGTCCAGGTTCGGTA-3′) and probe TR9096-P2-7680F (5′-6FAM-CGCTGCCTCATGGCAATTCG-BHQ1-3′) were used for the detection and quantification of ARPV and ARPV/ZIKV. In vitro transcribed RNA was used to generate standard curves. Primers ZIKV-1086F (5′-CCGCTGCCCAACACAAG-3′), ZIKV-1162cR (5′-CCACTAACGTTCTTTTGCAGACAT-3′) and probe ZIKV-1107-FAM (5′-6FAM-AGCCTACCTTGACAAGCAGTCAGACACTCAA-BHQ1-3′) were used for the detection and quantification of ZIKV as previously described [35 (link)].

For quantification of viral RNA copies in sera collected on D29, D36, D42 and D50, viral RNA was first extracted from 100 µL serum using NucleoSpin^® RNA Virus kit from Nagel-Macherey. A one-step TaqMan qRT-PCR was performed using the iTaq ^TM universal probes One-Step kit (BIO-RAD, Hercules, CA, USA) with specific FL13 primers located in ORF1: forward primer 5’-TGTTTCCCCACAGATGTTTCG-3’, reverse primer 5’-CCAGGATTTTGAGGCTTTTCC-3’ and fluorescent probe FAM 5’-CCCCGAGTCAGTATC-3’ TAMRA (Sigma-Aldrich, Merck). The FL13b RNA standard curve was made with 10-fold dilutions from 10¹ to 10⁵ TCID₅₀/mL, each spiked in control pig serum and extracted with the NucleoSpin^® RNA Virus kit. The qRT-PCR was performed with 2 µL of sample elution in 10 µL final mix and the cycling involved the following steps: reverse transcription at 50 °C for 10 min, denaturation at 95 °C for 1 min, amplification 40 cycles at 95 °C for 3 s and 60 °C for 30 s. TaqMan run of experimental samples contained 2 replicates, mock pig serum RNA and H20. The TCID₅₀ equivalent per mL (TCID₅₀ eq/mL) serum was calculated from the FL13 standard curve, and the limit of detection was estimated to be 100 TCID₅₀ eq/mL. The reactions were carried out in a CFX Connect^TM light cycler (BIO-RAD).

Total bone marrow RNA was obtained by flushing femurs with 1 ml of Trizol (Invitrogen). RNA was prepared according to the manufacturer’s specification. One-step quantitative reverse-transcription PCR was performed using 50 ng of total RNA and the iTaq^TM Universal Probes One-Step Kit (Bio Rad, Hercules, CA) with no template and no reverse transcriptase controls. mRNA expression is normalized to β-actin mRNA expression. Data was collected on a StepOnePlus^TM Real-Time PCR System (Thermo Fisher). The following quantitative PCR primer/probe sets were used:
TaqMan® Assays and Arrays:

Amplification of RNA extracted from tick samples and blood meal was done by RT-qPCR targeting the non-structural NS2A region of WNV genome, using iTaq^TM Universal Probes One Step Kit (BioRad Laboratory Inc., Munich, Germany) and CFX-96 Real-Time system (BioRad Laboratory Inc., Munich, Germany) as previously described [45 (link)]. The samples were tested in duplicates and, for the quantification of relative viral titres, tenfold-dilution series of WNV RNA were run in parallel as standards, each dilution point having three replicates (Table S3, Figures S2 and S3). Standards used in the RT-qPCR reaction were obtained from WNV viral stocks with a defined titre (TCID50/mL), by extracting 100 µL of viral aliquots with TRIZOL reagent, according to the manufacturers’ instructions.