Primers used for the nested amplification of the VZV immediate
early-63 (
IE-63) gene and the amplification of the humanβ
-actin gene have been previously described.
15 16 17 For
the first round of PCR amplification, 20% of the extracted DNA from
each sample analyzed was incubated in a 50-μl reaction using
conditions previously detailed,
13 14 except 2.5 mM
MgCl
2 and 50 pmol of each primer was used, and no[
32P]dATP was added. The first cycle consisted
of 3 minutes each at 95°C, 58°C, and 72°C followed by 35 cycles
at the same temperatures for 1 minute each. For the second round of PCR
amplification of the nested VZV PCR, 20% of the first amplification
reaction was used as template in similar reaction conditions using 50
pmol of each internal primer. The first cycle of the nested
amplification consisted of 3 minutes each at 95°C and 72°C followed
by 35 cycles at the same temperatures for 1 minute each. The β-actin
PCR used similar reaction conditions, except 20 pmol of each primer and
only 2% of the extracted DNA was used. The cycle parameters of theβ
-actin PCR were 3 minutes each at 95°C, 61°C, and 72°C,
followed by 35 cycles at the same temperatures for 45 seconds each.
Control DNA templates are described in the text and included plasmids
pVZV-
EcoE and pVZV-
EcoB; and viral DNA isolated
from cell cultures infected with herpes simplex virus (HSV) type 1
(strains KOS and 17+syn), HSV type 2 (strain HG52), or murine
cytomegalovirus (MCMV). Control PCR reactions included approximately 1
ng viral DNA. Analysis of all PCR reactions included electrophoresis of
20% of the reaction on 1.8% agarose/0.5× Tris-borate-EDTA (TBE)
electrophoresis gels containing 0.5 μg/ml ethidium bromide.