21.15109/ARP/GH1AYK Máté, Dóra0009-0001-7200-4823(Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest)Varga-Kugler, Renáta0000-0002-0093-5418(Ceva-Phylaxia Ltd.)Kaszab, Eszter0000-0002-5750-1872(Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest)Károlyi, Henrik Fülöp0009-0007-5035-7701(National Laboratory of Virology, Szentágothai Research Centre, University of Pécs)Görföl, Tamás0000-0002-1910-4024(National Laboratory of Virology, Szentágothai Research Centre, University of Pécs)Kemenesi, Gábor0000-0001-9775-3065(National Laboratory of Virology, Szentágothai Research Centre, University of Pécs)Igriczi, Barbara0000-0001-7372-3144(Department of Pathology, University of Veterinary Medicine Budapest)Balka, Gyula0000-0003-2742-173X(Department of Pathology, University of Veterinary Medicine Budapest)Domán, Marianna0000-0002-1823-9971(HUN-REN Veterinary Medical Research Institute)Bálint, Ádám0000-0002-2494-1097(Vetcontrol Ltd.)Zádori, Zoltán(HUN-REN Veterinary Medical Research Institute)Fehér, Enikő0000-0001-7778-9116(Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest) ARP 2026 hdl:21.15109/ARP/GH1AYK/6IDME2hdl:21.15109/ARP/GH1AYK/BZ9LC3 The rapid evolution of coronaviruses (CoVs) requires researchers to develop specific yet broad-spectrum detection methods to monitor their constant genomic changes. The goal of the present study was to establish a current pan-coronavirus RT-PCR system capable of detecting a wide variety of CoVs and useful for the investigation of virus diversity and host spectrum. For optimization, one-step and two-step nested RT-PCRs with three RT enzymes were examined, amplifying a ~600 bp long product of the RNA-dependent RNA polymerase. As templates, the in vitro transcribed RNA of ten pathogenic CoVs (SARS-CoV, SARS-CoV-2, NL-63, OC43, feline CoV, porcine epidemic diarrhea virus or PEDV, transmissible gastroenteritis virus or TGEV, canine CoV, bat CoV, and infectious bronchitis virus) were applied instead of the often-used DNA standards. A limit of detection of 5–50 copies/reaction was achieved with a random hexamer-primed two-step RT-PCR and a touchdown cycling profile, representing a lower detection limit and higher specificity compared to previously published primer sets. Swine origin pooled samples (n = 121), collected from apparently healthy herds in Hungary, were tested with the novel RT-PCR system. Sequences of porcine respiratory CoV/TGEV and porcine hemagglutinating encephalomyelitis virus were identified in 24 oral fluid and nasal swab pools, demonstrating the circulation of these viruses in this country, as well as the suitability of the new PCR for their detection. The results highlighted the importance of adequate RT enzyme selection and the use of RNase inhibitors in sample preparation and conservation. Máté, Dóra(Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest)