# Simultaneous detection of respiratory virus RNA on environmental surfaces in a university setting by a sensitive Surface 3-Step PCR platform

## TL;DR

This study uses a new PCR platform to detect respiratory viruses on university surfaces, showing higher contamination in smaller classrooms.

## Contribution

The study introduces a novel, sensitive Surface 3-Step PCR platform for simultaneous detection of multiple respiratory viruses on environmental surfaces.

## Key findings

- 62 out of 400 surface swabs tested positive for viral RNA, with positivity rates decreasing over time.
- Contamination was highest in small classrooms compared to medium and large ones.
- The platform achieved 98% valid results with high sensitivity for low viral RNA copy numbers.

## Abstract

Most respiratory viruses like SARS-CoV-2 spread through aerosols and fomites, remaining viable in the air and on surfaces. 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 48–49; T2, the extraordinary exam session: January 2024, weeks 2–4; T3, the start of the 2nd semester: February 2024, weeks 8–9) 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.

The online version contains supplementary material available at 10.1038/s41598-025-22296-5.