Summary

This white paper evaluates 35% hydrogen peroxide vapour (HPV) as a solution for RNA/DNA contamination control in qPCR laboratories. Based on experimental data from three real-world laboratory environments, it explores methodology, acceptance criteria, results and practical implications. HPV demonstrated significant reductions in environmental contamination, offering a rapid, automated alternative to manual deep cleaning. However, variability in results highlights areas for further research.

Background

PCR-based diagnostics are highly sensitive, amplifying trace nucleic acids to detectable levels. While this sensitivity is essential for accurate diagnostics, it also creates vulnerability to contamination. Even minimal RNA/DNA fragments can lead to false positives, compromising forensic investigations or clinical decisions.

Traditional cleaning methods can be resource-intensive and inconsistent, particularly in complex laboratory environments. HPV, widely used for aseptic pharmaceutical environments, offers a promising oxidative mechanism to degrade nucleic acids.

The rationale for this study was to determine whether HPV could effectively degrade RNA/DNA fragments to levels that help prevent amplification in PCR reactions. This approach aligns with the industry need for rapid, automated and validated decontamination processes that help minimise downtime and operational disruption.

Study Design

Three real-world qPCR laboratories with high levels of background RNA/DNA contamination were identified. The laboratories measured 54 m³, 76 m³ and 121 m³.

Thirty swabbing locations per laboratory were sampled before and after HPV exposure using the laboratories’ standard protocols. Six-log Geobacillus stearothermophilus biological indicators were used to confirm sporicidal efficacy. The targeted level of reduction in positive amplification sites was 90%. HPV cycle parameters were based on standard Bioquell Rapid Biodecontamination Service (RBDS) settings. Swabbing was performed using sterile viscose swabs across 10 cm × 10 cm areas, processed via qPCR using COVID-19 and pMA primers. Quantification was based on Cq values and standard curves to calculate RNA/DNA copy numbers.

For the FAM channel, which detects PCT amplicon, Cq ≤ 38 indicated the presence of amplicon contamination. Cq values above this threshold were considered negligible. For the HEX channel, which detects IEC amplicon, Cq ≤ 35 indicated the presence of amplicon contamination. Cq values above this threshold were considered negligible.

Results and Discussion

Average concentration reductions ranged from 82.7% to 86.0%. Variability was observed across surface types and contamination levels, suggesting that RNA/DNA presentation and material characteristics can influence the level of amplicon reduction. High-concentration samples showed negligible reduction, indicating limitations in HPV’s ability to degrade dense or occluded nucleic acid deposits. Cycle parameters were not optimised for nucleic acid degradation, presenting opportunities for improvement. Anomalies included persistent contamination on sinks and undersides of benches, highlighting the need for tailored cycles and further investigation into surface-material interactions.

Despite these limitations, HPV consistently reduced contamination in complex laboratory settings within a practical timeframe. The process also achieved validated six-log sporicidal kill, reinforcing its robustness for aseptic environments.

The variability in results suggests a relationship between contamination presentation and HPV efficacy. While some surfaces achieved significant decontamination, others retained measurable contamination despite similar exposure conditions. This indicates that factors such as surface porosity, airflow patterns and condensation dynamics may influence outcomes.

Operationally, HPV offers significant advantages over manual cleaning. Traditional deep cleans require full laboratory shutdowns, extensive labour and risk of equipment damage. In contrast, HPV cycles can be completed in approximately 4.5 hours with minimal disruption, helping preserve laboratory throughput and reduce contamination risk. These benefits align with regulatory expectations for contamination control and support continuous improvement initiatives in molecular diagnostics.

Practical Implications

HPV provides a scalable, automated solution for contamination control in PCR laboratories. Its compatibility with complex setups reduces the need for equipment removal, making it relevant for high-throughput environments.

However, laboratories must validate HPV cycles for nucleic acid degradation, as current parameters are typically optimised for sporicidal activity.

Recommendations

• Evaluate the implementation of HPV cycles as part of routine contamination control in PCR laboratories.
• Optimise cycle parameters for nucleic acid degradation, including hydrogen peroxide injection and dwell time.
• Investigate the role of RNA/DNA presentation and surface type in HPV efficacy.

Conclusion

35% HPV technology provides an effective, scalable approach for reducing RNA/DNA contamination in molecular laboratories. While variability and limitations warrant further research, HPV offers speed, automation and practical value for contamination control, offering speed, automation and compliance with industry standards. Its integration into routine laboratory protocols can potentially enhance operational efficiency and data integrity.

Future research should explore optimization of HPV cycles specifically for nucleic acid degradation, as current cycles are designed for sporicidal activity. Extending dwell times or adjusting peroxide concentration may improve efficacy against dense contamination. Understanding the interaction between HPV and various surface materials will also help laboratories predict and mitigate residual contamination risks.

Used as part of a validated contamination control strategy, HPV can help enhance operational efficiency and support data integrity in molecular diagnostic environments.

This document offers general guidance only and does not replace facility-specific risk assessments, regulatory requirements, validation protocols or manufacturer instructions.