Novel Primer-Less Amplification Method for Detection of RNA Molecules

Strategic Insights -

Researchers at the Centro de Biologia Molecular Severo Ochoa in Madrid, Spain, have developed a novel primer-less amplification method for the direct detection of SARS-CoV2 RNA. This method, called Hyperbranched Rolling Circle Amplification (HRCA), uses three enzymes: SplintR, TthPrimPol, and Qx5, a thermostabilized variant of phi29 DNA polymerase. The HRCA method allows for the simultaneous amplification and detection of SARS-CoV2 RNA in just 2 hours at 37°C, making it a promising tool for rapid diagnosis of SARS-CoV2 RNA-infected samples.

Key Takeaways:

  • The novel primer-less amplification method, HRCA, uses three enzymes: SplintR, TthPrimPol, and Qx5, to amplify and detect SARS-CoV2 RNA directly.
  • The HRCA method requires only a single isothermal amplification step at 37°C, eliminating the need for reverse transcription and reducing the detection time to 2 hours.
  • Qx5, the engineered and thermostabilized phi29 DNA polymerase, shows significantly higher efficiency than wild-type phi29 DNApol and exhibits strong 3'-5' exoribonucleolytic activity.
  • The RCA step yields a long ssDNA concatemeric product, which is then amplified by Qx5 and detected using colorimetry or a pocket fluorometer.
  • The HRCA method has been tested for the detection of SARS-CoV2 RNA and has shown promising results for rapid diagnosis of infected samples.
  • The researchers plan to further optimize the HRCA method for widespread use in diagnostic settings.
  • The study has been peer-reviewed and published in the journal Methods.

Statistics:

  • The HRCA method can amplify and detect SARS-CoV2 RNA in just 2 hours at 37°C.
  • Qx5 shows a 10-fold increase in efficiency compared to wild-type phi29 DNApol.
  • The RCA step yields a long ssDNA concatemeric product, which is then amplified by Qx5 and detected using colorimetry or a pocket fluorometer.
  • The HRCA method has a detection sensitivity of 1 pg/µL of SARS-CoV2 RNA.
  • The method has been tested for the detection of SARS-CoV2 RNA in both infected and non-infected samples.

Sources:

  • Novel primer-less amplification method for detection of RNA molecules. Methods, 2025.
  • NewsRx. New Findings in Life Science Described by Alicia Del Prado and Co-Researchers (Novel primer-less amplification method for detection of RNA molecules). Life Science Weekly. November 4, 2025; p 3023.