Related Experiment Video
Updated: Aug 28, 2025
Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples
Published on: June 30, 2023
Efficiency and sensitivity optimization of a protocol to quantify indoor airborne SARS-CoV-2 levels
J Truyols-Vives1, K Stiliyanov-Atanasov1, E Sala-Llinàs2
1Molecular Biology and One Health Research Group (MolONE), Universitat de les Illes Balears (UIB), Palma, Spain.
Background:
Development of methodologies to quantify airborne micro-organisms is needed for the prevention and control of infections. It is difficult to conclude which is the most efficient and sensitive strategy to assess airborne SARS-CoV-2 RNA levels due to the disparity of results reported in clinical settings.
Aim:
To improve our previously reported protocol of measuring SARS-CoV-2 RNA levels, which was based on bioaerosol collection with a liquid impinger and RNA quantification with droplet digital polymerase chain reaction (ddPCR).
Methods:
Air samples were collected in COVID-19 patient rooms to assess efficiency and/or sensitivity of different air samplers, liquid collection media, and reverse transcriptases (RT).
Findings:
Mineral oil retains airborne RNA better than does hydrophilic media without impairing integrity. SARS-CoV-2 ORF1ab target was detected in 80% of the air samples using BioSampler with mineral oil. No significant differences in effectiveness were obtained with MD8 sampler equipped with gelatine membrane filters, but the SARS-CoV-2 copies/m3 air obtained with the latter were lower (28.4 ± 6.1 vs 9 ± 1.7). SuperScript II RT allows the detection of a single SARS-CoV-2 genome RNA molecule by ddPCR with high efficiency. This was the only RT that allowed the detection of SARS-CoV-2 N1 target in air samples.
Conclusion:
The collection efficiency and detection sensivity of a protocol to quantify SARS-CoV-2 RNA levels in indoor air has been improved in the present study. Such optimization is important to improve our understanding of the microbiological safety of indoor air.
Insights
Optimized air sampling using mineral oil and SuperScript II reverse transcriptase (RT) enhances detection of airborne SARS-CoV-2 RNA. This improved method increases sensitivity for assessing indoor air microbiological safety.
Area of Science:
- Environmental microbiology
- Molecular diagnostics
- Infectious disease control
Background:
- Quantifying airborne microorganisms is crucial for infection prevention.
- Assessing airborne SARS-CoV-2 RNA levels lacks a consistently efficient and sensitive strategy.
- Previous protocols for SARS-CoV-2 RNA measurement require improvement.
Purpose of the Study:
- To enhance a previously established protocol for measuring SARS-CoV-2 RNA in bioaerosols.
- To optimize methods for collecting and quantifying airborne SARS-CoV-2 RNA.
- To improve the sensitivity and efficiency of detecting SARS-CoV-2 RNA in indoor air.
Main Methods:
- Air samples were collected from COVID-19 patient rooms.
- Evaluated different air samplers, liquid collection media (mineral oil vs. hydrophilic), and reverse transcriptases (RT).
- Quantified SARS-CoV-2 RNA levels using droplet digital polymerase chain reaction (ddPCR).
Main Results:
- Mineral oil demonstrated superior airborne RNA retention compared to hydrophilic media.
- The BioSampler with mineral oil detected SARS-CoV-2 ORF1ab target in 80% of samples.
- SuperScript II RT enabled high-efficiency detection of single SARS-CoV-2 genome RNA molecules and was essential for detecting the N1 target.
Conclusions:
- The study successfully improved the collection efficiency and detection sensitivity of a protocol for quantifying airborne SARS-CoV-2 RNA.
- Optimized methods enhance the understanding of microbiological safety in indoor air environments.
- The refined protocol offers a more sensitive approach to monitoring airborne viral RNA.
