21.15109/ARP/GH1AYKMáté, DóraDóraMáté0009-0001-7200-4823Department of Microbiology and Infectious Diseases, University of Veterinary Medicine BudapestVarga-Kugler, RenátaRenátaVarga-Kugler0000-0002-0093-5418Ceva-Phylaxia Ltd.Kaszab, EszterEszterKaszab0000-0002-5750-1872Department of Microbiology and Infectious Diseases, University of Veterinary Medicine BudapestKárolyi, Henrik FülöpHenrik FülöpKárolyi0009-0007-5035-7701National Laboratory of Virology, Szentágothai Research Centre, University of PécsGörföl, TamásTamásGörföl0000-0002-1910-4024National Laboratory of Virology, Szentágothai Research Centre, University of PécsKemenesi, GáborGáborKemenesi0000-0001-9775-3065National Laboratory of Virology, Szentágothai Research Centre, University of PécsIgriczi, BarbaraBarbaraIgriczi0000-0001-7372-3144Department of Pathology, University of Veterinary Medicine BudapestBalka, GyulaGyulaBalka0000-0003-2742-173XDepartment of Pathology, University of Veterinary Medicine BudapestDomán, MariannaMariannaDomán0000-0002-1823-9971HUN-REN Veterinary Medical Research InstituteBálint, ÁdámÁdámBálint0000-0002-2494-1097Vetcontrol Ltd.Zádori, ZoltánZoltánZádori6602902322HUN-REN Veterinary Medical Research InstituteFehér, EnikőEnikőFehér0000-0001-7778-9116Department of Microbiology and Infectious Diseases, University of Veterinary Medicine BudapestARP2026Agricultural Sciencespan-coronavirusPCRmammalianavianhumanswinemolecular diagnosticsMáté, DóraDóraMátéDepartment of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest2026-02-232026-02-242020/202210.3390/ani160303587763283198text/plaintext/plain1.1CC BY-NC 4.0The 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.HungaryNational Research, Development, and Innovation OfficeFK154149National Research, Development, and Innovation OfficeFK137778National Research, Development, and Innovation OfficeRRF-2.3.1-21-2022-00001Ministry of Culture and Innovation of Hungary2024-2.1.1-EKÖP-2024-00018