Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA.

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Point-of-care (POC) diagnostics face challenges with RNA detection methods requiring specific temperature control.
• Existing enzymatic amplification and visualization techniques often necessitate heating, limiting their applicability in resource-limited settings.

Purpose of the Study:

• To develop a novel, isothermal RNA detection method that operates at room temperature.
• To create an integrated amplification and detection platform for sensitive RNA quantification suitable for POC applications.

Main Methods:

• Reverse transcription lesion-induced DNA amplification (RT-LIDA), an isothermal method utilizing T4 DNA ligase for RNA-templated DNA primer ligation.
• Toehold-mediated strand displacement for exponential amplification of complementary DNA (cDNA) via isothermal ligase chain reaction (LIDA).
• Room-temperature colorimetric detection of DNA amplicons using DNA-modified gold nanoparticles.

Main Results:

• RT-LIDA successfully amplified RNA targets at a stable 28°C, functioning within global room temperature ranges.
• The method achieved high sensitivity, detecting as little as ~100 amol of target RNA and differentiating it from total cellular RNA.
• Integrated amplification and colorimetric detection were demonstrated at room temperature, requiring no external instrumentation.

Conclusions:

• RT-LIDA provides a sensitive, isothermal RNA detection platform operating entirely at room temperature.
• The developed system significantly advances the potential for instrument-free point-of-care diagnostic tools.
• This technology offers a promising solution for accessible and rapid RNA detection in diverse settings.