Fluorescent RT-RAA for Diagnosing Epidemic Diarrhea in Pigs

Objective  A rapid fluorescence RT-RAA to detect porcine epidemic diarrhea virus (PEDV) was developed and tested for field application.

Methods Primers and probes were designed for the conserved region of PEDV-S gene fragment, and a standard plasmid was constructed. Through condition optimization followed by tests for assay specificity, sensitivity, and repeatability, a recombinant enzyme-mediated chain replacement nucleic acid amplification fluorescence RT-RAA for detecting PEDV was established.

Results  Under a constant temperature of 42 ℃ for 20m, the assay detected PEDV as positive, while negative on the porcine transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV) and other porcine viruses. It had a minimum detection limit of 4.43×10² standard plasmid copies·μL⁻¹, a reproducibility showing little difference between the standard plasmids of the same concentration, and a positive rate of 7.5% (3 out of 40) on PEDV specimens comparable to that obtained by RT-qPCR.

Conclusion  The newly developed fluorescence RT-RAA for PEDV detection was considered appropriate for rapid diagnosis of epidemic diarrhea in pigs.