Transscript probe one step rt ri enzyme mix

All real-time RT-PCR assays were performed on ABI Quant Studio 5 (Applied Biosystems, Foster City, CA, United States). Each reaction consisted of 2×PerfectStart Probe One-Step qPCR SuperMix (TransGen Biotech, Beijing, China) 12.5 μl, TransScript Probe One-Step RT/RI Enzyme Mix 0.5 μl, 1 μl of standard RNA, in addition to different volumes of primers (0.6, 0.8, 1.0, 1.2, 1.4, 1.6 μl, 10 μmol/L) and probe (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 μl, 10 μmol/L), which were used to optimize the assay and made up to 25 μl with the RNase-Free ddH₂O. The reaction condition was set as follows: 45°C for 5 min and 94 °C for 30 s, followed by 40 cycles of 94°C for 5 s and 55–62°C for 30 s, which were used to confirm the optimal amplification conditions. All optimization assays were performed in parallel with the same standard RNA as the template.

Real-time RT-PCR assay was used to detect the BRVA in the cattle fecal samples using primers and probe from a previously published protocol [18 (link)]. The reaction volume was 25 μL, which included 12.5 μL of 2 × PerfectStart™ Probe One-Step qPCR SuperMix (TransGen Biotech, Beijing, China), 0.5 μL of TransScript^@ Probe One-Step RT/RI Enzyme Mix, 0.5 μL of each primer and probe (10 μmol/L), 3 μL of RNA template and 7.5 μL of Nuclease free water. The reaction condition was 94 °C for 5 min, 94 °C for 30 s, 40 cycles of 94 °C for 5 s and 60 °C for 30 s. Real-time RT-PCR was performed using ABI Quant Studio 5 (Applied Biosystems, Foster City, California).