Superscript 3 one step kit

The total RNA of cells and mosquito body homogenates were isolated using TRIzol reagent (Invitrogen), according to the standard manufacturer's instructions. Viral RNA from mosquito saliva samples was extracted and purified using a Mag-Bind Viral RNA 96 kit (Omega). The yields and the quality of the RNA samples were determined by Nanodrop (Thermo). To determine the presence of the two USUV strains in co-infection samples, a one-step RT-PCR using a single pair of primers followed by enzymatic digestion of the DNA amplicon was performed. From the viral RNA, a 900 bp DNA fragment was synthesized and amplified using the Superscript III One-Step kit with Platinum Taq DNA polymerase (Invitrogen) and primers (Forward: 5'-GGATGTTGGTATGGAATGGAGATA-3'; Reverse: 5'-GTCGATTTGCCTGAAATGGTGT-3'), annealing to the viral NS1 to NS2A genes. Restriction enzyme digestion using DraI (NEB) was used to differentiate between USUV-NL and USUV-IT, as the DraI restriction site is only present in the USUV-NL amplicon. DNA band intensity was quantified using GelAnalyzer 19.1 software. Bands having a pixel count of less than 5% of the total pixel count for that specific sample were considered undigested products and therefore excluded from the analysis.

Quantitative PCR was performed using the Superscript III One Step kit (Invitrogen Corp) in a Roche Lightcycler 480 (Roche Diagnostics). Standard curves were generated for each primer set, and samples fitted to the linear portion of the curve. Additionally, PCR products were run on DNA gels to verify single products. Primer sequences used were described previously and were as follows: FXNFWD 5′- ATCTTCTCCATCCAGTGGACCT-3′ and FXNREV 5′- GCTGGGCATCAAGCATCTTTT-3′; ARE16223FWD 5′- CCTGCCGTACTCAGTCCTTC-3′ and ARE16223REV 5′- CCACTCGGCTGTACTGTCTG-3′; NQO1FWD 5′-CCCTTTTAGCCTTGGCACGAAA-3′ and NQO1REV 5′- TGCACCCAGGGAAGTGTGTTGTAT-3′; HO1FWD 5′ CCCTGCTGAGTAATCCTTTCCCGA-3′ and HO1REV 5′- ATGTCCCGACTCCAGACTCCA-3′ (67 (link)).

Viral extraction from the samples was performed with a PureLink™ Viral RNA/DNA Mini Kit (Invitrogen, USA) according to the manufacturer’s instructions. The full S1 segment (~1600 bp) was amplified with the IBVS1-F (5′-AACTCTGCACGCAAATTA-3′) and IBVS1-R (5′-TGTTGTCGCAAACAGGACC-3′) primers described by Zhang et al. [23 (link)].
The RT-PCR was performed using a SuperScript^® III One-Step kit (Invitrogen, USA) with the following parameters: 30 min at 55 °C, 2 min at 94 °C, 40 cycles of 15 s at 94 °C, 30 s at 49 °C, finally an extension step at 68 °C for 5 min was added. The RT-PCR products were visualized in an agarose gel then purified using a PureLink™ Quick Gel Extraction Kit according to the manufacturer’s instructions. Finally, an ABI 3730XL genetic analyzer was used in the sequencing process.
The alignment calculation for the nucleotide sequences was performed with Mafft 7.2 software [24 (link)] using the L-INS-i method. The nucleotide model substitution was then identified using Jmodeltest software v2.1.7 [25 (link)], and a maximum-likelihood tree was constructed with the online platform PhyML 3.0 [26 (link)] using the dataset previously published by Valastro et al. [14 (link)]. Finally, the amino-acid sequences were aligned using Bioedit v7.2.5 [27 ].