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.