Profiling RT-LAMP tolerance of sequence variation for SARS-CoV-2 RNA detection

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Technologies

Background:

• The COVID-19 pandemic demands scalable molecular diagnostics for SARS-CoV-2 detection.
• Emerging SARS-CoV-2 variants pose challenges to existing nucleic acid amplification tests.
• Reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers a rapid, cost-effective alternative to RT-qPCR but requires further validation.

Purpose of the Study:

• To investigate the impact of SARS-CoV-2 sequence mutations on RT-LAMP assay performance.
• To assess the robustness of RT-LAMP against primer region variations and deletions.
• To explore the use of molecular beacons for variant-specific detection using RT-LAMP.

Main Methods:

• Generated 523 single point mutation variants across LAMP primer regions for three SARS-CoV-2 assays.
• Performed over 4,500 RT-LAMP reactions to analyze amplification kinetics and sensitivity.
• Designed and tested primer sets targeting a specific short deletion, including those with multiple mismatches.

Main Results:

• Minimal impact on RT-LAMP amplification speed and no loss of detection sensitivity were observed for most single point mutations.
• RT-LAMP successfully amplified RNA with primers containing multiple consecutive mismatched bases, albeit with reduced performance.
• Molecular beacons enabled sensitive discrimination of variant RNA sequences with short deletions.

Conclusions:

• RT-LAMP is a robust molecular diagnostic technique capable of tolerating most mutations within primer regions.
• The method demonstrates significant potential for reliable viral RNA detection in the face of evolving SARS-CoV-2 variants.
• RT-LAMP combined with molecular beacons can be adapted for sensitive variant identification and molecular diagnostics outside clinical settings.