2.2 Swab collection, RNA extraction and real-time RT-PCR
Oropharyngeal and fecal swabs were collected from the cats at various time-points, to identify the period of viral shedding (Figure 1). All samples were obtained by means of 14.5-cm snappable sterile polystyrene/Dacron swabs (Deltalab, Barcelona, Spain). Immediately after sample collection, the swab tips were being placed in 2-mL microcentrifuge tubes containing 800 μL of guanidinium isothiocyanate-based “Lysis buffer I”. Lysates were subjected to a phenol-chloroform-based RNA extraction process coupled with silica column binding (Chaintoutis, Papadopoulou, Melidou, Papa, & Dovas, 2019).
All swabs obtained from the 3 cats at all time-points were tested for the presence of SARS-CoV-2 genome, via 2 separate real-time RT-PCR assays proposed as suitable for the laboratory diagnosis of COVID-19 in humans by international agencies. Specifically, the protocol proposed by the European Centre for Disease Prevention and Control (ECDC) for the amplification of the genomic region that encodes the E protein was used (Corman et al., 2020). Similarly, the N2 method targeting the genomic region that encodes the N protein, which is proposed by the Centers for Disease Control and Prevention (CDC) within the “CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel” was applied (Centers for Disease Control and Prevention (CDC), 2020). All oligonucleotides (primers and TaqMan probes) were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Both assays were performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Analysis of fluorescence data was performed using CFX Maestro™ software (version 4.1; Bio‐Rad Laboratories, Hercules, CA, USA). RNA extracts with a Ct value > 40 were considered as negative. Calibration curves were also generated for virus quantification. Virus titers were Log10-expressed as mean SARS-CoV-2 RNA copies per real-time RT-PCR-positive swab.