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).