Sybr green 1 reaction mix

Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green I Reaction Mix (Toyobo) in a Roche 480 Real Time Detection System (Roche, Basel, Switzerland). The primers used in the experiment were listed at Table 1. Each assay was carried out in triplicate under the following conditions: 1 min for activation at 95°C followed by 40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. The transcription levels of target genes were normalized to β-actin (reference gene) and calculated using the 2^−ΔΔCT method. The data are presented as the mean ± standard deviation.

To determine the effects of different reagents on RGNNV replication, the transcript of CP (fragment from 391–621 nn) was detected by qPCR. In brief, mock- or RGNNV-infected cells were collected, and the total RNA was extracted using an SV total RNA isolation system (Promega) according to the manufacturer’s instructions. The RNA was reverse transcribed using a ReverTra Ace qPCR RT Kit (TOYOBO). Amplification was examined using a SYBR Green I Reaction Mix (Toyobo) in an Applied Biosystems QuantStudio 5 Real Time Detection System (Thermofisher, United States). Each assay was carried out under the following cycling conditions: 95°C for 1 min for activation, followed by 40 cycles at 95°C for 15 s, 60°C, for 15 s, and 72°C for 45 s. The primers used in the experiment were those that were described previously (Zhang et al., 2019 (link)). The level of target gene expression normalized to β-actin was calculated using the 2^–ΔΔCT method. The data are representative of one representative experiment carried out in triplicate.

To determine the effects on different inhibitors on virus replication, viral gene transcriptions were detected by qRT-PCR. Total RNA from SGIV-infected cells were isolated using the cell total RNA isolation kit (Foregene) according to manufacturer’s instructions. The RNA was reverse transcribed using ReverTra Ace RT Kit (Toyobo). Amplification was examined using SYBR Green I Reaction Mix (Toyobo) in an Applied Biosystems QuantStudio 5 Real Time Detection System (Thermo Fisher). Each assay was carried out under the following cycling conditions: 95 °C for 5 min for activation, followed by 40 cycles at 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s. The primers used were listed in Table 1. The expression levels of target genes were standardized to β-actin and calculated with the 2^−△△CT method. The data were indicated as mean ± SD and shown from one representative experiment carried out in triplicate. Statistical significance was determined with Student’s t-test and the statistical significance was set at p < 0.05.