SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal...

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Assays

Background:

• Nucleic acid amplification tests (NAATs) are sensitive but not ideal for point-of-care use.
• Rapid antigen tests are user-friendly but lack diagnostic sensitivity and specificity.
• There is a need for rapid, sensitive, and user-friendly SARS-CoV-2 detection methods.

Purpose of the Study:

• To develop a one-pot, duplexed reverse transcriptase recombinase polymerase amplification (RT-RPA) assay for SARS-CoV-2 detection.
• To incorporate a full process control using MS2 bacteriophage for assay validation.
• To achieve rapid, sensitive, and visually readable results suitable for point-of-care applications.

Main Methods:

• Development of a one-pot, duplexed RT-RPA assay for simultaneous detection of SARS-CoV-2 and MS2 bacteriophage.
• Utilized real-time fluorescence or lateral flow assay (LFA) for result readout.
• Incorporated MS2 bacteriophage as an internal control to monitor lysis, reverse transcription, and amplification.

Main Results:

• Achieved analytical sensitivity of 50 copies per reaction for SARS-CoV-2 detection.
• Demonstrated successful operation of all assay steps, including lysis, reverse transcription, and amplification, via the MS2 control.
• Obtained results in under 25 minutes with minimal instrumentation, including a visual LFA readout.

Conclusions:

• The developed SARS-CoV-2 RT-RPA assay offers a sensitive and rapid diagnostic method.
• The integrated MS2 bacteriophage control enhances test reliability by ruling out false negatives.
• This assay presents a promising alternative for point-of-care COVID-19 diagnostics.