Inter-laboratory comparison and additional characterization:
The optimized the triplex assay along with the gB assay were transferred to Kansas State University Veterinary Diagnostic Laboratory (KSU-VDL) for inter-laboratory comparison using a negative cohort study comprised of negative clinical samples collected from U.S. swine herds. A few known-negative clinical samples were spiked with synthetic PRV-specific DNA fragments (gBlocks) and tested on the triplex assay to mimic positive clinical situations.
For the negative cohort study, KSU-VDL used 400 known PRV-negative clinical samples collected from healthy pigs in the U.S. As positive controls, 22 clinical samples (serum, oral fluids, and fecal swabs) were spiked with PRV classical or PRV variant gBlocks. The assay was also evaluated at the U.S. Department of Agriculture’s National Veterinary Services Laboratories (NVSL) in Ames, Iowa, for its ability to detect a second PRV variant strain, HeN1 (An et al., 2013) using archived vaccine challenge study samples (58 nasal swabs and 58 oral swabs), which were collected from seven pigs vaccinated with a USDA licensed, commercially available PRV DIVA vaccine and five unvaccinated pigs, all of which had been challenged with the PRV HeN1 (NVSL, unpublished data).