Validation of a standardized quantitative RT-qPCR workflow for detection and quantification of airborne respiratory viruses using gelatin membranes as sampling matrix

: Airborne microbiological monitoring is increasingly relevant for identifying and quantifying viral pathogens; however, standardized analytical workflows for respiratory viruses recovered from environmental matrices remain limited. This study reports the full analytical validation of a quantitative RT-qPCR workflow for the simultaneous detection and quantification of SARS-CoV-2, influenza A, influenza B, and RSV A/B from air samples collected on gelatin membrane filters. The protocol optimizes RNA extraction and amplification from this challenging matrix and provides a standardized procedure suitable for reproducible laboratory implementation. All validation parameters-amplification efficiency, linearity, recovery, matrix interference, limit of detection (LOD), limit of quantification (LOQ), repeatability, and reproducibility-were assessed in compliance with ISO 20395 and MIQE guidelines and met all predefined acceptance criteria. The workflow showed excellent linearity (R2 ≥ 0.99 for all targets), high amplification efficiency (92-103%), robust recovery (81.98-92.28%), and no significant matrix interference (±10%). LODs ranged from 5 to 9 copies/reaction, and LOQs from 8 to 10.5 copies/reaction. Precision assessments demonstrated acceptable intra- and inter-test variability (CV% ≤ 35%, SD < 0.19). Overall, this validated and standards-compliant RT-qPCR workflow provides a reliable, sensitive, and reproducible analytical tool for laboratory quantification of airborne respiratory viruses collected on gelatin membrane filters, supporting standardized virological analysis of aerosol samples.