Development of a novel reverse transcription droplet digital PCR assay for the sensitive detection of Senecavirus A

In pigs, Senecavirus A (SVA) causes a vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. Sensitive and specific detection of SVA is critical for controlling this emerging disease. In this study, a novel reverse transcription droplet digital PCR (RT-ddPCR) assay, targeting the conserved viral polymerase 3D gene, was established for the detection of SVA. This assay exhibited good linearity, repeatability and reproducibility, and maintained linearity at extremely low concentrations of SVA nucleic acid templates. The detection limit of RT-ddPCR was 1.53 ± 0.22 copies of SVA RNA per reaction (n = 8), and the assay showed approximately 10-fold greater sensitivity than a reverse transcription real-time PCR (RT-rPCR) assay. Moreover, specificity analysis showed that the RT-ddPCR for SVA had no cross-reactivity with other important swine pathogens. In clinical diagnosis of 134 pig serum and tissue samples, 26 and 21 samples were identified as positive by RT-ddPCR and RT-rPCR, respectively. The overall agreement between the two assays was 96.27% (129/134). Further linear regression analysis showed a significant correlation between the RT-ddPCR and RT-rPCR assays with an R² value of 0.9761. Our results indicate that the RT-ddPCR assay is a robust diagnostic tool for the sensitive detection of SVA, even in samples with a low viral load.