Virus detection via real-time PCR
Prior to real-time PCR analysis, nucleic acids from soil samples were
extracted using the DNeasy PowerSoil kit (Qiagen, Hilden, Germany)
according to the manufacturer’s recommendations. Subsequently, nucleic
acids were analyzed using a published real-time PCR assay targeting the
ASFV p72 gene (Tignon et al., 2011) in combination with an internal
control based on beta actin (Toussaint et al., 2007) on a CFX96
real-time cycler (Bio-Rad Laboratories, Hercules, USA). PCR was
performed with the QuantiTect Multiplex PCR Kit (Qiagen, Hilden,
Germany) in a total volume of 25 µL. Using a dilution series of an 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) and the exact number of DNA molecules was calculated with an
online tool
(http://www.molbiol.edu.ru/eng/scripts/01
07.html).