Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2...

Area of Science:

• Environmental microbiology
• Molecular diagnostics
• Public health technology

Background:

• Wastewater-based surveillance is crucial for monitoring infectious diseases like COVID-19.
• Existing methods for SARS-CoV-2 detection in wastewater lack point-of-use capabilities.
• A sensitive and specific detection tool for SARS-CoV-2 in wastewater is needed.

Purpose of the Study:

• To develop a portable, point-of-use device for SARS-CoV-2 detection in wastewater.
• To achieve sensitive and specific detection using CRISPR/Cas12a and RT-LAMP.
• To enable semiquantitative analysis of SARS-CoV-2 in wastewater samples.

Main Methods:

• Developed a paper device integrating CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP).
• Utilized three primer sets and guide RNAs targeting N, E, and S genes of SARS-CoV-2.
• Employed CRISPR/Cas12a trans-cleavage activity with fluorescence or lateral flow probes for visualization.

Main Results:

• Achieved limits of detection (LODs) of 25, 310, and 10 copies/mL for N, E, and S genes, respectively.
• Enabled semiquantitative analysis within a range of 0 to 310 copies/mL.
• Demonstrated 97.7% sensitivity and 82% semiquantitative accuracy in blind wastewater samples.

Conclusions:

• The developed paper device is a promising point-of-use tool for SARS-CoV-2 detection in wastewater.
• This represents the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater using differential LODs.
• The device enhances wastewater-based surveillance capabilities for infectious disease outbreaks.