Magmax viral pathogen extraction kit

The swab samples were processed, and real-time RT-PCR was undertaken, following the standardized protocols for SARSR-CoV-2 detection in animal samples at Penn State’s ADL. RNA was extracted from 400 µL of swab samples, using a KingFisher Flex machine (ThermoFisher Scientific, Waltham, MA, USA) and a MagMAX Viral/Pathogen extraction kit (ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. The presence of SARS-CoV-2 viral RNA was further tested, using the OPTI Medical SARS-CoV-2 RT-PCR kit, which is a highly sensitive assay that targets the N gene [26 (link),27 (link)]. The RT-PCR assays were carried out on an ABI 7500 Fast instrument (ThermoFisher Scientific, Waltham, MA, USA). The internal control RNase P was utilized, to confirm that the samples were not contaminated with human tissue or fluids during harvesting or processing. The samples were also tested using a TaqPath kit (ThermoFisher Scientific, Waltham, MA, USA), which targets the SARS-CoV-2 ORF1ab, N gene, and S gene [27 (link),28 (link)] as the first screen for Omicron, which typically presents as an S gene drop out in these assays [28 (link)].

RNA was extracted from RPLNs by adding 3 mL of Universal Transport Medium (UTM, Copan) to a Whirl-Pak bag containing the tissue and placing the bag in the stomacher on a high setting for 120 s. Liquid was recovered and centrifuged at 3,000 rpm for 5 min to pellet cellular debris. Then 400 μL of the RPLN tissue homogenate supernatant was used for viral RNA extraction with a KingFisher Flex machine (ThermoFisher Scientific) with the MagMAX Viral/Pathogen extraction kit (ThermoFisher Scientific) following the manufacturer’s instructions.