Homebrew reagents for low-cost RT-LAMP

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) is a sensitive and specific method for detecting SARS-CoV-2.
• High reagent costs and cold chain requirements limit RT-LAMP accessibility, especially in low-income regions.
• Homebrew enzyme and buffer preparation offers a potential solution to these limitations and reagent shortages.

Purpose of the Study:

• To describe the low-cost production of Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase for local RT-LAMP implementation.
• To optimize reagent concentrations for improved RT-LAMP performance, including reduced time to threshold and increased ON/OFF range.
• To validate the performance of in-house prepared reagents for SARS-CoV-2 detection using clinical samples.

Main Methods:

• Production of key enzymes (Moloney murine leukemia virus reverse transcriptase and BstLF DNA polymerase) for RT-LAMP.
• Optimization of enzyme concentrations to enhance reaction kinetics and sensitivity.
• Validation of in-house RT-LAMP assays against RT-PCR using clinical nasopharyngeal samples.

Main Results:

• In-house produced reagents demonstrated comparable performance to commercial kits.
• Optimized concentrations led to reduced time to threshold and improved ON/OFF range.
• High agreement was observed between in-house RT-LAMP and RT-PCR for SARS-CoV-2 detection in clinical samples.
• An order of magnitude reduction in cost was achieved through in-house preparation.

Conclusions:

• Local production of essential enzymes enables cost-effective and accessible RT-LAMP assays for SARS-CoV-2 detection.
• The developed protocols and DNA resources facilitate replication of these low-cost diagnostics globally.
• This work supports the development of open-source, low-cost diagnostics, promoting technological autonomy and distributed viral surveillance capabilities.