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).