Rapid on-site PEDV detection using homogeneous fluorescence resonance energy transfer-based ELISA

The porcine epidemic diarrhea virus (PEDV) is a major pathogen of swine enteric diseases. Various etiology and serological methods have been employed for PEDV detection, but most of their applications are limited to laboratories. To extend PEDV detection to on-site applications, we design a homogeneous fluorescence resonance energy transfer (FRET)-based enzyme-linked immunosorbent assay (ELISA). Both donor and acceptor fluorescence microspheres modified PEDV antibodies can be linked only to the occurrence of PEDV antigen, thus generating FRET signals, which can be collected by our designed portable FRET immunoassay station (FRETIS). Verified by standard samples, FRET immunoassay reached high sensitivity with a detection limit as TCID50 (median tissue culture infective dose) of 10/mL, which is 10 times more sensitive than colloidal gold test strips; and verified by clinical samples, it was also proved with high accuracy, good selectivity, and repeatability. More importantly, FRET immunoassay could detect PEDV in a 96-well plate in 35 min with only one step of incubation without any further washing steps using field-portable devices and field-operable procedures, well supporting on-site applications. Considering these advantages, this reported FRET immunoassay provides a promising way for multi-sample on-site PEDV detection and can be potentially used in the swine industry.