Gotaq probe 1 step rt qpcr system kit

Nucleic acids extraction was performed using 200 µL of clarified cell supernatant medium with the EZ1 mini virus 2.0Kit and the EZ1 Biorobot (both from Qiagen) according to the manufacturer’s instructions. Relative quantification of viral RNA was performed using the GoTaq probe 1-step RT-qPCR system kit (Promega). The mixture (final volume: 20 µl) contained 10 µl of GoTaq probe qPCR Master Mix, 0.5 µL of each primer (10 µM working solution were used), 0.2 µl of probe (10 µM working solution were used), 0.5 µl of Go script RT mix, 0.3 µl of nuclease-free water and 8 µl of extracted nucleic acids. Assays were performed using the CFX96 Touch real-time PCR machine (Bio-Rad) with the following conditions: 50 °C for 15 min, 95 °C for 2 min, followed by 45 cycles of 95 °C for 15 s, 60 °C for 40 s. Data collection occurred during the 60 °C step. The amount of viral RNA was calculated from standard curves using synthetic RNA. Primers used are described in the Supplementary Table S3 or are previously described in^{18 (link),21 (link)}.

Anti-HEV antibody and HEV antigen were detected by commercial ELISA kits (Wantai, Beijing, China) according to the manufacturer’s instructions. Signal-to-cut-off (S/CO) values were calculated and values >1 were considered positive.
Total RNA was extracted from 100 μL of serum or fecal suspensions prepared as above using TRIzol reagent (Invitrogen, Burlington, ON, Canada) following a standard instruction. The RNA was then amplified by RT-nPCR for partial ORF2 genome on an automatic PCR system (Applied Biosystems ProFlex PCR, USA) and HEV viral load was determined using a commercial one-step real-time quantitative PCR assay (GoTaq® Probe 1-step RT-qPCR System Kit, Promega, Wisconsin, USA), as previously described [32 (link)].

The presence of MAYV in the RNA samples was detected by RT-qPCR with the AriaMX system (Agilent Technologies, Santa Clara, CA, USA) using the GoTaq^® Probe 1-Step RT-qPCR System Kit (Promega, Madison, WI, USA) according to the manufacturer's recommendations. The set of primers/probes amplifying the actin gene of Mansonia spp. were used as an endogenous control of RT-qPCR. We used gBlocks (Integrated DNA Technologies, Coralville, IA, USA), recognized by the primer and probe pairs designed to amplify the MAYV nonstructural polyprotein gene (GenBank: MK956954.1) or the actin gene of Mansonia spp. (GenBank: GQ981455.1) as a positive control. The viral genome copy number was calculated by plotting the quantification cycle (C_q) values reported in RT-qPCR against the standard curve [13 (link)] in which the threshold for detection of endogenous control (actin) and MAYV was determined, and C_q values above the detection threshold were considered negative. The gBlocks, probes, and primer sequences used in this study are listed in Additional file 1: Table S1 and Additional file 2: Table S2, respectively. The detection limit for the actin gene was five copies with a C_q value of 34.30 (Additional file 3: Fig. S1). For MAYV, the detection limit was 10 copies with a C_q value of 34.98 [13 (link)].