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been challenged with the PRV HeN1 (NVSL, unpublished data).