FCV RT-qPCR Detection and Quantification

Prior to TNA extraction, oropharyngeal cytobrush samples stored in DNA/RNA shield (Zymo Research) were thoroughly mixed and incubated at 40 °C for 10 min. TNA was extracted from 100 µL EDTA anticoagulated blood or 200 µL RNA/DNA shield (Zymo Research) using the MagNa Pure LC (Roche Diagnostics AG) and the MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics AG) according to the manufacturer’s instructions. Positive and negative RT-qPCR controls were run in parallel. Two different real-time RT-qPCR assays (FCV RT-qPCR S1 and S2) were used to detect FCV, as previously described [7 (link)]. For a semiquantitative analysis of FCV viral loads in cytobrush and blood samples, FCV RNA standards were run in parallel.