RNA Extraction and RT-PCR
NP and R swabs were resuspended by vortexing in 300µL of PBS. Total RNA
was extracted from 140 µL of supernatant using either the robotic
workstation ‘QIAcube’ and the QIAamp Viral RNA kit (Qiagen; reference:
52906) or robotic method based on magnetic beads (KingFisher with the
MagVet Universal Isolation kit) according to the manufacturer’s
instructions and the protocols classically used in our laboratory for
other viruses such as Foot-and-Mouth Disease, Bluetongue or West Nile
viruses. Five µL of eluted RNA were used for the RT-qPCR assay using the
commercial kit Bio-T kit® Covid-19 (BioSellal - Dardilly
-France)) targeting the E gene.
At the same time, all the rectal swabs were tested by RT-qPCR targeting
the feline coronavirus (FCoV) using the protocol previously described
(D’Orengiani et al, 2015).
SARS-CoV-2 confirmatory tests were carried out in the OIE collaborating
Centre in Institut Pasteur using the real-time duplex RT-PCR (targeting
two regions of the RdRP gene) developed by the French National Reference
Centre for Respiratory Viruses and the real-time RT-qPCR (targeting the
E gene) from the Charité protocol (see WHO Coronavirus disease COVID-19
technical guidance: Laboratory testing for 2019-nCoV in humans,
available fromhttps://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf ).