Ion AmpliSeq SARS-CoV-2 of cat with COVID-19 symptomatic owner
Total RNA extracted from rectal swab sample was extracted using a QIAmp viral RNA mini kit (Qiagen, Germany). Quantitation of total extracted RNA was performed by fluorimetry using Qubit (Life Technologies, Thermo Fisher Scientific, CA, USA). Reverse transcription was performed using SuperScriptTM VILOTM cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific, CA, USA). Multiplex PCR was conducted using amplification mastermix for each sample with 5X Ion AmpliSeqTM HiFi Mix (Ion AmpliSeq Library Kit Plus, Ion Torrent, Life Technologies, CA, USA). DNA library was prepared using Ion AmpliSeqTM SARS-CoV-2 Research Panel (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturers instructions. PCR products were purified using an ProNex Size- Selective Purification System (Promega, Wisconsin, USA). Pooled libraries were sequenced on Ion Torrent S5 technology (Thermo Fisher Scientific, Massachusetts, USA) with size-selection with approximately 200 bp fragments.