Development of a VP2-based real-time fluorescent reverse transcription recombinase-aided amplification assay to...

Senecavirus A (SVA), a newly emergent picornavirus correlated with sudden neonatal mortality and vesicular lesions in pigs, has had a considerable impact on the global pig farming industry. Timely and dependable detection of SVA is helpful in preventing the further spread of this pathogenic virus. In the current study, a real-time fluorescent reverse transcription recombinase-aided amplification (rRT-RAA) assay, which targets the most conserved region within the VP2 gene of SVA, was developed and evaluated for SVA detection. The detection limit for this assay was tested to be 1.185 50% tissue culture infective dose (TCID₅₀ ) of SVA RNA per reaction at a 95% confidence interval, which is comparable to that of a previously published rRT-PCR assay for SVA. The testing results of the rRT-RAA assay were very reproducible and repeatable, with inter- and intra-assay coefficient of variation values less than 7.0%. In addition, the established rRT-RAA assay displayed excellent specificity for SVA detection without cross-reaction with other clinically important swine pathogenic viruses. The diagnostic performance of rRT-RAA was evaluated using 189 clinical swine samples, which were detected in parallel using the reference rRT-PCR assay. The results showed that 146 and 151 samples tested positive for SVA by rRT-RAA and rRT-PCR, respectively. The overall agreement between both assays was 97.4% (184/189) with a kappa value of 0.927 (p < .001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R²   = 0.9192, p < .0001). Taken together, our newly established rRT-RAA assay is a powerful and time-saving diagnostic tool for SVA detection in clinical samples.