Novel primer-less amplification method for detection of RNA molecules.

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Accurate and rapid detection of SARS-CoV-2 RNA is crucial for pandemic control.
• Existing methods often require complex steps like reverse transcription, increasing time and cost.
• There is a need for simplified, sensitive, and isothermal nucleic acid amplification techniques.

Purpose of the Study:

• To develop a simplified, isothermal amplification method for direct SARS-CoV-2 RNA detection.
• To demonstrate the efficacy of Hyperbranched Rolling Circle Amplification (HRCA) for viral RNA detection without reverse transcription.
• To utilize engineered enzymes for enhanced efficiency and primer generation.

Main Methods:

• Isothermal amplification using Hyperbranched Rolling Circle Amplification (HRCA).
• Enzymes used: SplintR for probe ligation, TthPrimPol for primer synthesis, and engineered phi29 DNA polymerase (Qx5) for amplification.
• Direct detection of viral RNA by utilizing its 3' polyA tail as a primer after trimming by Qx5.

Main Results:

• Successful isothermal and primer-less amplification of specific padlock probes for SARS-CoV-2 RNA detection.
• Significant DNA amplification achieved in 2 hours at 37°C.
• Demonstrated ability to differentiate between SARS-CoV-2 infected and non-infected samples via colorimetry or fluorometry.

Conclusions:

• The developed HRCA method offers a simplified, rapid, and sensitive approach for SARS-CoV-2 RNA detection.
• This technique eliminates the need for reverse transcription, streamlining the diagnostic process.
• The method shows promise for quick and accessible point-of-care diagnosis of viral infections.