Development and optimization of a single-tube, triplex real-time qPCR assay.
A triplex assay that can differentiate highly virulent PRV variant strains from classical strains was successfully designed targeting the US2- US9 intergenic region. This region is deleted in Bartha-K61 and Bucharest PRV strains which are commonly used in commercial PRV marker vaccines; therefore, the assay can be used to differentiate animals vaccinated with these vaccines from those infected with wild-type PRV strains (DIVA assay). For the assay development, genomic DNA was extracted from PK-15 cells infected with PRV classical strain Bristol, PRV Variant strain JS-2012, and PRV gE- deleted DIVA vaccine strain Bartha-K61. Prior to use for assay characterization, the sequence identity of these three virus strains was confirmed by PCR amplification of the region flanking the gE deleted region of the Bartha-K61 strain using forward primer 5’-GTACCGGCGTCGATGATGAT-3” and reverse primer 5’-GCCCAGGATCCACAGGTG-3’, followed by Sanger sequencing.
The triplex assay conditions were optimized using the synthetic gene fragments (gBlocks) resembling the target regions of PRV classical and variant virus genomes and the β-actin gene (data not shown) according to the cycling conditions recommended for the TaqMan FAST Virus 1-Step Master Mix kit. The optimized triplex assay was able to accurately detect and differentiate the PRV classical strains Bristol, Shope and Becker and the variant strain JS-2012. The triplex assay did not detect any of the closely related herpesviruses or other high consequence swine viral pathogens tested, including ASF, CSF, and FMD viruses (Table 2). As expected, the triplex assay was unable to detect the gE-deleted PRV Bartha-K61 Vaccine strain and the PRV Bucharest vaccine strain. The gB assay, in contrast, detected all tested PRV strains, confirming the presence of PRV viral DNA in these samples.
Once the triplex assay was optimized, the standard curves of the assay were generated and PCR efficiency was determined by plotting Ct value against log copy number from ten-fold serial dilutions of PRV plasmid with net copy number of 7.34x1011 for both classical and variant strains of PRV (Figure 2). The assay values with Ct values >40 were not plotted on the graph. The regression line equation for PRV Variant (FAM channel) was y = -3.3563x + 39.416 with a correlation coefficient (R2) of 0.9941. The regression line equation for PRV Classical (HEX channel) was y = -3.5375x + 41.126 with a correlation coefficient (R2) of 0.9907. The PCR efficiencies were determined to be 98.59% and 91.73% for variant and classical strain targets, respectively.
To determine the limit of detection of the assay, PRV plasmid was diluted ten-fold to extinction. The triplex assay was able to detect as low as 0.86 copies for PRV Variant (FAM) and 1.86 copies for PRV Classical (HEX) targets.
The repeatability of the triplex assay was evaluated by intra-assay and inter-assay variability testing. The coefficient of variation (CV) within each replicate was determined as a percentage of the ratio of standard deviation and mean of Ct values from FAM (specific for variant strain) and HEX (specific for classical strain) channels. Inter-assay variability testing was done with the triplex run three times over three days with one replicate for each dilution of PRV Bristol and JS-2012 viruses. Intra-assay variability was calculated with 20 replicates in a single run using the 10-2 dilution of PRV JS-2012 and Bristol viruses. All variability testing was performed on the Applied Biosystems 7500 FAST real-time PCR machine. The triplex assay demonstrated good repeatability for the diluted samples of PRV JS-2012 and PRV Bristol with the inter-assay ranging from 0.52 ≤ CV ≤ 3.64 and the intra-assay CV ranging from 1.74 ≤ CV ≤ 2.02 for both strains. The diluted samples of both PRV strains were run on three different real-time PCR instruments including the Light Cycler 480, Applied Biosystems 7500, and Bio-Rad CFX 96. The results were consistent across the real-time PCR platforms tested (data not shown).