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

Figure 5

Optimizing reaction conditions for BCoV RT-RAA-LFD assay. (a) Optimization of primer and probe concentration. Primer concentrations were tested at five levels 2, 4, 6, 8, and 10 μM as were probe concentrations. The optimal concentration for both primer and probe was found to be 2 μM. RT-RAA-LFD experiments conducted with higher concentrations of primer and probe yielded false-positive results. (b) Optimization of reaction temperature. Temperatures were set at six gradients 33, 35, 37, 39, 41, and 43°C. The optimal temperature was determined to be 35°C, with observable results after 12.5 min of reaction time. When the temperature exceeded 39°C, the negative control group exhibited specific amplification. Additionally, as the temperature increased, the color intensity of the T line on the test strip for the negative control group became more pronounced, while that of the positive test strip gradually lightened. (c) Optimization of reaction time. Reaction times were tested at seven intervals 5, 7.5, 10, 12.5, 15, 17.5, and 20 min. Bands began to appear at 7.5 min and were clearest at 12.5 min.

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