Inter-laboratory comparison and additional characterization:
Inter-laboratory comparison of the triplex assay was performed at the KSU Veterinary Diagnostic Laboratory and the NVSL in Ames, Iowa. At the KSU VDL, 440 negative samples including oral fluids, serum, feces, lung, tissue and nasal swabs collected from the U.S. commercial swine herd were tested on the triplex real-time PCR and the gB real-time PCR (data not shown). All clinical samples were negative for both classical and variant PRV nucleic acid, as expected. Fluorescent signal for Q670 (endogenous internal control, β-actin) was detected on all samples, with Ct values ranging from 18.73 to 35.85, confirming efficient DNA extraction and absence of PCR inhibitors in the samples (data not shown).
As positive controls, 11 negative serum samples and one negative oral fluid sample were spiked with the PRV classical gBlock, nucleic acid was extracted and tested on the triplex assay. All of these spiked samples were detected in the HEX and Q670 channels, and no detection was reported in the FAM channel of the triplex assay. Similarly, seven negative serum samples, four negative fecal swab samples and one negative oral fluid samples were spiked with the PRV variant gBlock, nucleic acid was extracted and tested on the triplex assay. All of these spiked samples were detected in the FAM and Q670 channels, and no detection was reported in the HEX channel of the triplex assay.
The triplex assay was further evaluated at the NVSL using archived oral and nasal swabs collected from a vaccine challenge experiment conducted in 2015 (NVSL, unpublished data). In the experiment, seven weaned piglets were vaccinated with a USDA licensed, commercial gE/gI deleted PRV marker vaccine; and five piglets were used as non-vaccinated controls. All pigs were challenged with the highly virulent PRV variant strain HeN1. A total of 116 nasal and oral swabs from this experiment were tested on the triplex assay (24 samples each from -14, 0, 2 and 5-days post challenge and 20 samples each from 8-days post challenge).The assay detected variant PRV starting 2 days post challenge in all the samples. With the exception of two samples which were near the limit of detection on the triplex assay, the results were in agreement with the NVSL-run gB real-time PCR (Ma et al. 2008; Zanella et al. 2012). The triplex assay did not detect any classical PRV genomes in any of these samples tested (data not shown). As positive controls for the triplex assay, known negative nasal and oral swab samples were spiked with gBlocks representing PRV variant, PRV classical, and beta-actin.