Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification for In Situ Detection of SARS-CoV-2 in a...

Area of Science:

• Molecular Diagnostics
• Virology
• Environmental Monitoring

Background:

• The COVID-19 pandemic accelerated the need for rapid airborne infectious agent detection.
• Current airborne virus detection methods face limitations in intervention timeliness.
• Autonomous identification systems for airborne pathogens are under exploration.

Purpose of the Study:

• To explore reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detecting Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) on membrane filters.
• To optimize RT-LAMP assay conditions for micro-air-filtration of airborne viruses.
• To enhance the sensitivity and timeliness of airborne virus detection.

Main Methods:

• Development of a one-pot RT-LAMP assay for SARS-CoV-2 detection.
• Utilizing membrane filters compatible with micro-air-filtration systems.
• Employing a design of experiments statistical framework to optimize additive composition for RT-LAMP on filters.
• Conducting liquid spike-in experiments with SARS-CoV-2 and fluorescence detection.

Main Results:

• Established optimal additive composition for RT-LAMP on membrane filters.
• Demonstrated reliable detection of 0.10 50% tissue culture infectious dose (TCID50) of SARS-CoV-2 per reaction (3600 E-gene copies) using single-pot RT-LAMP on glass fiber filters.
• Achieved an order of magnitude greater sensitivity compared to conventional RT-LAMP assays.

Conclusions:

• Single-pot RT-LAMP on membrane filters is a sensitive and reliable method for detecting airborne SARS-CoV-2.
• This approach offers a promising advancement in molecular diagnostics for airborne virus exposure risk assessment.
• The optimized assay has the potential to improve intervention timeliness in airborne pathogen detection.