Easyscript one step rt pcr supermix

According to the manufacturer’s instructions, the total RNA was extracted from the specimens using the RNeasy Mini Kit (Qiagen, Germany). The extracted RNA was used to detect the presence of NNV by RT-PCR using EasyScript® one-step RT-PCR Super Mix (Transgen Biotech, China) according to the manufacturer’s instructions and primers targeting the RNA2 gene, VNNF:5′-ACA CTG GAG TTT GAA ATT CA-3′ and VNNR:5′-GTC TTG TTG AAG TTG TCC CA-3′, which amplified 605 bp (Dalla Valle et al. 2000 (link)). In brief, a total reaction volume of 20 μl contains 10 μl of 2 × ES One-Step Reaction Mix, 0.4 μl of EasyScript One-Step Enzyme Mix, 0.4 μl of each primer (20 pmol), 3.8 μl of RNase-free water, and 5 μl of template RNA. The optimized thermal cycling conditions for one-step RT-PCR were as follows: a cDNA synthesis step at 45 °C for 30 min, PCR amplification as an initial denaturation cycle at 94 °C for 5 min, and 35 cycles of denaturation at 94 °C for 30 s, annealing at 49 °C for 30 s, and extension at 72 °C for 1 min. The final extension step was performed at 72 °C for 10 min. The PCR products were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide at 0.5 μg/ml concentration.

One-step RT-PCR was used to detect the existing FMDV serotype in the extracted RNA using serotype-specific PCR amplification of the VP1 protein genomic region. The used primer sets were amplifying 814, 1124, and 666 bp of the 1D variable gene of serotypes A, O, and SAT2, respectively, as described previously [21 (link)]. The reaction was carried out using the EasyScript^® one-step RT-PCR SuperMix (TransGen, Beijing, China) according to manufacturer instructions. The thermal conditions were adjusted as described previously [14 (link)]. The PCR products were documented via UV rays after electrophoresis on 1.2% agarose gel in a Tris-acetate EDTA buffer stained with ethidium bromide for 45 min [8 (link)].

HBsAg from HepG2 cell lysate and supernatant was detected with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Autobio, Zhengzhou, China). HBV DNA from cell lysate and supernatant was measured by a quantitative fluorescent diagnostic kit (Sansure Biotech, Changsha, China). The tests were all carried out according to the manufacturer’s protocols.
Intracellular total RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany), and quantified by NanoDrop (Thermo Scientific, Waltham, MA, USA). We performed reverse transcription-qPCR (RT-qPCR) assay to detect HBV total RNA and pregenomic RNA (pgRNA). The reverse transcription was carried out using EasyScript One-Step RT-PCR SuperMix (TransGen Biotech, Beijing, China). The primer sequences for RT-qPCR were shown in Table S1. Ribosomal protein S11 mRNA was used to normalize RT-qPCR results for HBV transcripts.