Nucleic acid extraction and real-time PCR
Prior to real-time PCR analysis, viral nucleic acids from all samples
(inoculum, blind passage, virus suspension) were extracted using the
NucleoMag VET kit (Macherey-Nagel, Düren, Germany) on the automated
KingFisher 96 flex platform (Thermo Fisher Scientific, Schwerte,
Germany) according to the manufacturer’s recommendations. Subsequently,
nucleic acids were analyzed using a published real-time PCR (King et
al., 2003) in combination with an internal control based on an EGFP
detection system (Hoffmann et al., 2006) on a Biorad CFX real-time
cycler (Bio-Rad Laboratories, Hercules, USA). For each real-time PCR, a
quantification cycle (Cq) value was determined according to the PCR
cycle number at which the fluorescence of the reaction crosses a value
that is statistically higher than the background which is determined by
the Biorad CFX software. Using a dilution series of an in-house ASFV DNA
standard, the genome copies in the respective samples were determined.
For generation of the ASFV standard, DNA from an ASFV “Armenia08” PBMC
culture supernatant was extracted using the QIAamp Viral RNA Mini Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
recommendations. Subsequently, the DNA concentration was determined by
spectrophotometry using a Nanodrop 2000c (Thermo Fisher Scientific,
Schwerte, Germany) and the exact number of DNA molecules was calculated
using an online tool
(http://www.molbiol.edu.ru/eng/scripts/01
07.html).