Safeview classic

Transcription assay in vitro was conducted using Riboprobe Systems (catalog no. P1440, Promega, WI, USA) following the protocol provided by the manufacturer. The active reagent was prepared by mixing the pGEM express template with the reaction mixture containing 5× optimized buffer, DTT, ribonuclease inhibitor, ribonucleoside triphosphates, and T7 polymerase. Transcripts containing two bands of 2346 and 1065 bases were analyzed by agarose electrophoresis. Each reaction was mixed with serially diluted DPRs in PBS150 at final concentrations of 0, 2.5, 5, 7.5, and 10 μM. For SOS experiments, GR30 was mixed with various concentrations of SOS before subjected to the mixed reagent. The volume of GR30/SOS mixture was one-tenth to the mixed reagent. Mixtures were incubated at 37°C for 2 hours and loaded into a 1% agarose gel for electrophoresis. The gel was premixed with a nucleic acid dye, SafeView Classic (Applied Biological Materials, Richmond, BC, Canada), and images were captured by an ImageQuant LAS 4000 imaging system (GE Healthcare Life Sciences, IL, USA).

Allele-specific PCR was performed using the forward primer CacytbF (GGG TAT AGG TTT CCT GGG TTA TG) and the reverse primer S-mmc (ATA AGG TTA GTA ATA ACT GTT GCC C) for the amplification from sensitive isolates and R-mmc (ATA AGG TTA GTA ATA ACT GTT GCC G) for the amplification from resistant isolates (Kim et al., 2019 ). The primers were designed to specifically amplify a single point mutation at 143 amino acid position. A 20 μl PCR mixture containing 4 μl master mix (EzPCR 5× PCR master mix, Elpis Biotech), 1 μl of each primer (forward and reverse), 2 μl gDNA, and 12 μl SDW was prepared. The amplification was performed in a thermo-cycler (MiniAmp Plus, Thermo Fisher Scientific, Waltham, MA, USA) with an initial denaturation at 95°C for 4 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension 72°C for 1 min 20 s, and a final extension at 72°C for 5 min. To confirm the amplification of the genes by PCR, 1.5% agarose gel was made with 0.5× TBE buffer, and safe view classic (Applied Biological Materials Inc., Richmond, BC, Canada) was added at 5 μl per 100 ml of agarose solution. The PCR products were separated by electrophoresis for 40 min at 100 V and the gel was viewed on a UV-transilluminator.

The presence of PRRSV, PCV2, PM, HPS, APP, MHP, MHR, and SS in OF samples were determined by using reverse-transcriptase polymerase chain reaction (RT-PCR) or standard polymerase chain reaction (PCR) assays. For DNA/RNA extraction, samples were processed by using the Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol. After DNA/RNA extraction, each sample underwent RT-PCR (Maxime RT-PCR PreMix Kit; iNtRON Biotechnology) or PCR (Maxime PCR PreMix Kit; iNtRON Biotechnology) according to the properties of each pathogen. The primer sequences used in this study are listed in Table 1. To detect pathogens, RT-PCR, nested PCR, or PCR was carried out according to previously described protocols. In case of PRRSV, RT-PCR and nested PCR were performed sequentially with nested PCR used as a confirmatory test. The references describing the corresponding protocols for each pathogen are listed in Table 1.
The amplicons were separated by performing electrophoresis with 2.0% agarose gels and staining with DNA dye (SafeView Classic; Applied Biological Materials, Canada) and visualized under UV light. The sizes of the PCR products were determined by comparison with a DNA ladder.