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The present study aims to detect simultaneously by a multiplex molecular approach (Surface 3-step PCR platform), the presence of the three major co-circulating respiratory viruses (SARS-CoV-2, Flu A\/B and RSV A\/B) from inert surface samples in non-healthcare environments on a university setting in Central Italy. In total, 400 environmental surface swabs were collected during the study period in a three time point longitudinal program (T1, the end of the first semester: November-December 2023, weeks 48\u201349; T2, the extraordinary exam session: January 2024, weeks 2\u20134; T3, the start of the 2nd semester: February 2024, weeks 8\u20139) among which 62 (16%) were positive for viral RNA and with the positive rate that dropped from 20% (25\/125) to 8% (10\/130). The frequency of environmental contamination was higher in small classrooms (30\/135, 22%) than in medium (15\/105, 14%) and large (13\/115, 11%) ones. Here, we describe the use of a novel rapid and sensitive combined multistep molecular platform involving two process controls, one synthetic RNA added directly to the sample and one endogenous human, able to detect low copy numbers of viral RNA targets in high-touch surfaces, with high sensitivity (98% of valid results). This study shows the potential as an effective solution to apply targeted interventions to prevent the spread of the airborne infections within the university community.<\/jats:p>","DOI":"10.1038\/s41598-025-22296-5","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T09:19:36Z","timestamp":1762161576000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Simultaneous detection of respiratory virus RNA on environmental surfaces in a university setting by a sensitive Surface 3-Step PCR platform"],"prefix":"10.1038","volume":"15","author":[{"given":"Chiara","family":"Orlandi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulia","family":"Amagliani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giorgio","family":"Brandi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asja","family":"Conti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuditta Fiorella","family":"Schiavano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Casabianca","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,3]]},"reference":[{"key":"22296_CR1","doi-asserted-by":"publisher","unstructured":"Folgueira, L., Moral, N., Pascual, C. & Delgado, R. 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