Gelred nucleic acid gel staining

To confirm their equine origin and to screen for EcPV2 infection, proliferating early-passage cells were subjected to DNA extraction using QIAamp DNA Minikit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer’s instructions. PCR reactions were performed from 1 μL DNA aliquots using (i) horse specific GAPDH primers (ecGAPDHi forward primer: 5′-ATC CGG AGT CTT CCA CTC CA-3′, ecGAPDHi reverse primer: 5′-GTC GGA GGG TTA ACC ACA GG-3′) and (ii) EcPV2-specific primers for amplification of a 395 bp region within the E6 gene (18). Reaction mixtures of 25 μL were prepared according to the manufacturer’s instructions (REDTaq ReadyMIX, PCR Reaction mix; Sigma-Aldrich, Merck & Cie, Schaffhausen, Switzerland) and then subjected to a denaturation step of 94 °C for 3 min, followed by 40 amplification cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s per cycle. Amplicons were separated by 1% TAE-gel electrophoresis and visualized by GelRed^® Nucleic Acid Gel Staining (Biotium, Inc. Fremont, CA, USA).