Extraction-Free Colorimetric RT-LAMP Detection of SARS-CoV-2 in Saliva

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Technologies

Background:

• The COVID-19 pandemic underscored the need for rapid, affordable pathogen detection methods.
• Current standard RT-qPCR for SARS-CoV-2 requires lengthy nucleic acid extraction.
• Isothermal amplification methods offer potential alternatives for faster diagnostics.

Purpose of the Study:

• To develop and evaluate a novel, extraction-free colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection in saliva.
• To assess the sensitivity and specificity of the developed calcein RT-LAMP method compared to commercial kits and RT-qPCR.

Main Methods:

• A calcein-based, extraction-free RT-LAMP protocol was optimized for saliva samples.
• Samples were subjected to a brief heat treatment followed by isothermal amplification at 63°C.
• Results were assessed visually via color change (orange to green) or fluorescence.

Main Results:

• The developed calcein RT-LAMP method demonstrated 90% sensitivity and 100% specificity compared to RT-qPCR for saliva samples with Ct ≤ 34.
• The assay showed improved sensitivity and specificity over a commercial colorimetric RT-LAMP kit.
• The method achieved comparable performance to a commercial fluorescent RT-LAMP kit without requiring a fluorescence reader.

Conclusions:

• The calcein RT-LAMP assay provides a simple, rapid, and cost-effective diagnostic tool for SARS-CoV-2 detection in saliva.
• Its visual readout and extraction-free nature make it suitable for widespread population screening.
• This method presents a viable alternative to RT-qPCR, especially in resource-limited settings.