Development of Rapid Isothermal Detection Methods Real-Time Fluorescence and Lateral Flow Reverse Transcription Recombinase-Aided Amplification Assay for Bovine Coronavirus

Figure 4

Optimization of reaction conditions for real-time RT-RAA experiments with BCoV. (a) Primer pairs screening, three pairs of upstream primers, and three pairs of downstream primers were designed to form primer pairs F1/R1, F1/R2, F1/R3, F2/R1, F2/R2, F2/R3, F3/R1, F3/R2, and F3/R3. DNAzyme-free water was used as a negative control. The primer screening revealed that the F2/R3 pair had the highest amplification efficiency. (b) Primer volume optimization: The primer volume was tested at five different gradients 1.6, 1.8, 2.0, 2.2, and 2.4 μL with the optimal volume determined to be 2.2 μL. (c) Probe volume optimization: The probe volume was also tested at five different gradients 0.2, 0.4, 0.6, 0.8, and 1.0 μL with the optimal volume identified as 0.8 μL. (d) Reaction temperature optimization: The reaction temperature was evaluated at five different gradients 33, 35, 37, 39, and 41°C with the optimal temperature determined to be 39°C. The results could be observed after a 20 min reaction period.

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