SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and...

Area of Science:

• Molecular Biology
• Virology
• Diagnostics

Background:

• The SARS-CoV-2 pandemic necessitates expanded diagnostic capabilities for safe economic reopening.
• Current testing methods face limitations in speed, sensitivity, and accessibility.
• Rapid and reliable diagnostics are crucial for controlling viral spread.

Purpose of the Study:

• To develop a sensitive and rapid diagnostic assay for SARS-CoV-2 detection.
• To create an efficient sample preparation protocol compatible with the assay.
• To enhance the accessibility and scalability of SARS-CoV-2 testing.

Main Methods:

• Development of a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay.
• Optimization of a rapid viral inactivation and nuclease degradation protocol.
• Integration of a compatible RNA purification method to maximize sensitivity.
• Utilized a colorimetric readout for results within 30 minutes.

Main Results:

• The RT-LAMP assay achieved a sensitivity of 50 viral RNA copies/µL with rapid inactivation alone.
• The combined inactivation and purification protocol increased sensitivity to 1 viral RNA copy/µL.
• The assay demonstrated compatibility with existing reagents and provided results in under 30 minutes.
• The developed pipeline is inexpensive and uses readily available materials.

Conclusions:

• A simple, sensitive, and rapid RT-LAMP assay pipeline for SARS-CoV-2 detection has been established.
• This method significantly enhances diagnostic sensitivity and sample stability.
• The assay's affordability and compatibility expand testing capabilities and settings, aiding pandemic control efforts.