Development of a Crystal Digital RT-PCR for the Detection of Atypical Porcine Pestivirus

SIMPLE SUMMARY: Atypical porcine pestivirus (APPV), a newly discovered virus, is associated with the type A-II congenital tremor (CT) in neonatal piglets. Here, the specific primers and probe were designed, the reaction conditions were optimized, and a crystal digital RT-PCR (cdRT-PCR) and real-time quantitative RT-PCR (qRT-PCR) for the accurate detection of APPV were successfully established. Both assays were demonstrated to be highly sensitive, strong specific, and excellently repeatable. The sensitivity of the cdRT-PCR was 100 times higher than that of the qRT-PCR. The 60 clinical tissue samples were tested for APPV, showing 23.33% positivity by the qRT-PCR and 25% positivity by the cdRT-PCR, with a coincidence rate of 98.33%. The results illustrate that both established methods are useful tools for detection of APPV. ABSTRACT: Atypical porcine pestivirus (APPV), a newly discovered virus, is associated with the type A-II congenital tremor (CT) in neonatal piglets. APPV distributes throughout the world and causes certain economic losses to the swine industry. The specific primers and probe were designed targeting the 5′ untranslated region (UTR) of APPV to amplify a 90 bp fragment, and the recombinant standard plasmid was constructed. After optimizing the concentrations of primers and probe, annealing temperature, and reaction cycles, a crystal digital RT-PCR (cdRT-PCR) and real-time quantitative RT-PCR (qRT-PCR) were successfully established. The results showed that the standard curves of the qRT-PCR and the cdRT-PCR had R(2) values of 0.999 and 0.9998, respectively. Both methods could specifically detect APPV, and no amplification signal was obtained from other swine viruses. The limit of detection (LOD) of the cdRT-PCR was 0.1 copies/µL, and that of the qRT-PCR was 10 copies/µL. The intra-assay and inter-assay coefficients of variation of repeatability and reproducibility were less than 0.90% for the qRT-PCR and less than 5.27% for the cdRT-PCR. The 60 clinical tissue samples were analyzed using both methods, and the positivity rates of APPV were 23.33% by the qRT-PCR and 25% by the cdRT-PCR, with a coincidence rate of 98.33%. The results indicated that the cdRT-PCR and the qRT-PCR developed here are highly specific, sensitive methods for the rapid and accurate detection of APPV.