Onestep ahead rt pcr kit

Primers for nested RT-PCR, which amplify a 332 bp product from the HEV open reading frame 1 (ORF1) were designed by Johne et al. (2010 (link)). RNA from the HEV isolate 47832c (Johne et al. 2014 (link)) was used as positive control. For the first RT-PCR with a total reaction volume of 25 µl, 5 µl of template was amplified using the OneStep Ahead RT-PCR Kit (QIAGEN, Germany). Cycling profile included the following settings: 10 min at 50 °C, 5 min at 95 °C, 40 cycles of 10 s at 95 °C, 10 s at 55 °C, 10 s at 72 °C and 2 min at 72 °C. The second nested PCR was performed with 2 µl template from the first RT-PCR. The Taq DNA Polymerase Kit (QIAGEN, Germany) was used according to protocol in a total reaction volume of 50 µl. Primer concentrations were 0.3 µM and cycling conditions were the following: 3 min at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at 60 °C, 1 min at 72 °C and 10 min at 72 °C.
PCR fragments were separated by gel electrophoresis on 1.5% agarose gels in 1 × TBE buffer with 10 µl of 10,000×g GelRed staining (Biotium, Germany) per 100 ml agarose solution. Loading buffer (Thermo Scientific, Germany) was mixed with the PCR products and gels were run for 50 min at 90 V. Low range DNA ladder (5 µl) was used as a size marker (Thermo Scientific, Germany).

To amplify either the 414-to-583 RBD region or the 1-to-250 region of the spike gene from SARS-CoV-2 RNA samples, we used both one-step RT-PCR and two-step (cDNA synthesis followed by separate PCR amplification) reaction designs with a commercial OneStep Ahead RT-PCR kit (Qiagen) or IEH in-house RT, PCR, and RT-PCR kits according to manufacturer’s guidelines. Both kits and conditions yielded identical results. All primers used for amplification are listed in Table S5. The first round of PCR (either RT-PCR on RNA or PCR on cDNA) consisted of 40 cycles of 10 s at 95°C, 15 s at 57°C, and 40 s at 72°C. The product was then diluted 1:50 with sterile water, and a second round of PCR was performed for 15 cycles under the same conditions by using T7-tailed nested primers to obtain a single pure product. Sanger sequencing of the amplified region was performed from both ends using standard T7Promoter and T7Terminator primers by Eton Bioscience, Inc. Sequences were analyzed using BioEdit 7.2 and MEGA 7 software. Sequences were compared to the reference Wuhan-Hu-1 sequence (NC_045512), and unique sequences were assigned allele numbers (see the supplemental alleles file).

Total RNA was isolated by QIAzol method and reverse transcribed by OneStep Ahead RT-PCR Kit (Qiagen). All predesigned primers were purchased from Qiagen. mRNA level of TNF-α (tumor necrosis factor), IL-1β (interleukin 1 beta), MIP-2 (macrophage inflammatory protein-2), MCP1 (CD46), MAP1lc3 (microtubule-associated protein 1 light chain 3), VPS11 (vacuolar protein sorting-associated protein 11), or β-actin was detected by real-time PCR. The fold changes were determined by relative quantification method as described earlier [29 (link)].