PRV triplex qPCR assay development and optimization:
Three separate qPCR assays (classical PRV RT-PCR, variant PRV qPCR and
β-Actin qPCR) were first developed and later combined into a triplex
assay. The triplex assay was optimized on the Applied Biosystems 7500
Real-Time PCR System using detection channels for FAM (absorbance at
495nm, emission at 520nm), HEX (absorbance at 535nm, emission at 556nm),
and Q670 (absorbance at 647-670 nm). The optimized PCR reaction (20 µl)
contains 5.5 µL sterile PCR grade water, 5 µL 4X TaqMan FAST Virus
1-Step Master Mix, 1 µL of PRV forward and 1 µL PRV reverse primers (0.5
µM final), 0.5 µL PRV variant probe (0.25 µM final), 0.5 µL PRV
classical probe (0.25 µM final), 2 µL of β-Actin forward and 2 µL of
β-Actin reverse primers (1 µM final), 0.5 µL of β-Actin probe (0.2 µM
final) and 2 µL extracted nucleic acid from the sample. The optimized
triplex assay conditions are: 50ºC for 5 minutes, 95ºC
for 20 seconds, and 45 cycles of 95ºC for 3 seconds and 60ºC for 30
seconds. All primers and probe working stocks were maintained at 10 µM
concentration. The primers and probe for the gB qPCR assay was adapted
from Ma et al, (2008) and the forward and reverse primers were used at
0.4 µl per reaction (0.4 µM final) with the probe at 0.4 µl per reaction
(0.2 µM final) with the stock solutions being 20 µM for primers and 10
µM for the probe. The same PCR cycling conditions were used for the
triplex and the gB assays.