Primers and Probes:
Primers and probes to detect and differentiate classical and variant PRV strains were designed using Geneious v10.1.1 (Biomatters Inc. Newark, NJ, USA) bioinformatics software sequence analysis tool. To identify the target regions for the primers and probes, an alignment of 34 PRV sequences was compiled, consisting of 12 full-length and six partial classical strains, as well as 13 full-length and three partial strains of variant PRV. The sequence data for these viruses were downloaded from the NIAID Virus Pathogen Resource (ViPR - http://www.viprbrc.org) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The six partial sequences were included as they were used in the design of the triplex assay recently reported by Meng et al., 2016. Once the sequences were aligned, genomic regions showing multiple mutations or insertions between PRV classical and variant strains were identified. Of those targeted regions, the untranslated region between the US9 gene and the US2 gene was selected (from 127,349bp to 127,485bp nucleotides in JS-2012 PRV strain) as the preferred region for primer/probe design. Within this region, a 21-nucleotide insertion was identified in all variant strain sequences available, and it was used as the target for the PRV US9/US2 UTR variant probe. Downstream of the insertion, we identified a region that shows 13 nucleotide polymorphisms between classical and variant PRV strains. The PRV US9/US2 UTR classical probe was designed to capture 10 out of the 13 polymorphisms observed in this region. It was also observed that the targeted region is deleted in Bartha-K61 and Bucharest strains that are commonly used in commercial PRV glycoprotein E (gE)-deleted marker vaccines (Figure 1).
Two primers, PRV US9/US2 UTR RRT Forward (5’-ACACAGCAGCCTTCCT-3’) and PRV US9/US2 UTR RRT Reverse (5’-GCGTGACCACGGTGA-3’) were designed to amplify a 137bp long PCR product from variant PRV strains and a 116bp PCR product from classical strains. In order to differentiate classical and variant PRV strains, two strain-specific probes, PRV US9/US2 UTR classical and PRV US9/US2 UTR variant, were designed (Table 1A). As the internal control, primers and a probe specific for β-Actin (β-Actin 1036R 831FP, β-Actin 1036RP and β-Actin-probe-880) were used (Moniwa et al., 2007). For detection of all PRV strains, primers and probes from a widely used, highly sensitive and specific PRV gB real-time PCR assay were employed (Ma et al., 2008). To facilitate the triplex assay development, determine specificity, and to generate positive controls for the assay, synthetic gene fragments (gBlocks) of the target regions of PRV classical and variant genomes and the β-actin gene were synthesized and procured from Integrated DNA Technologies, Inc. (Coralville, Iowa) (Table 1B).