Novel primer-less amplification method for detection of RNA molecules
Year: 2025
Type: article
Abstract: • Novel Multiple Displacement Amplification methodology for detection of RNA targets that uses specific padlock probes in the absence of externally-added DNA primers. • A new engineered and thermostabilized phi29 DNA polymerase variant (Qx5) whose potent 3′-5′ exoribonucleolytic activity allows target RNA to to be used as primer of the RCA step. • The concatemeric ssDNA product from RCA is exponentially amplified by combining multiple priming by Tth PrimPol and subsequent Qx5 DNA polymerization activity, thus promoting hyperbranched amplification and boosting sensitivity of detection of the RNA target. As shown here, isothermal and primer-less amplification of specific padlock probes allows direct detection of SARS-CoV2 RNA without needing for a reverse transcription step. This simplified method of Hyperbranched Rolling Circle Amplification (HRCA) only requires three enzymes: SplintR (to ligate a specific padlock probe to its circular form, only when viral RNA is present), the unique DNA primase Tth PrimPol (to generate de novo DNA primers), and an engineered and thermostabilized variant of bacteriophage phi29 DNA polymerase (phi29 DNApol), named Qx5. Qx5 is significantly more efficient than wild-type phi29 DNApol and shows an unexpectedly strong 3′-5′ exoribonucleolytic activity capable of trimming the 3′ polyA tail of the RNA target, thus enabling it as a primer for Qx5 to start Rolling Circle Amplification (RCA) of a nearby circularized padlock. The RCA step, that yields a long ssDNA concatemeric product (RCA product, RCP) is coupled to a second isothermal amplification step, assisted by Tth PrimPol by synthesizing DNA primers on the RCP, that triggers exponential HRCA of the padlock sequence, catalyzed by Qx5. As a proof of concept, the application of this method for detection of SARS-CoV2 RNA rendered a significant amount of DNA in just 2 h at 37 °C, that can be easily evidenced by colorimetry, or even quantitated with a pocket fluorometer, and served for a quick diagnosis of SARS-CoV2 RNA infected versus non-infected samples.
Source: Methods
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,600
Funders: Fundación Ramón Areces, Ministerio de Ciencia, Innovación y Universidades, Centro de Biología Molecular Severo Ochoa, European Commission, Ministerio de Ciencia e Innovación, Fundación Bancaria Caixa d'Estalvis i Pensions de Barcelona, European Regional Development Fund, Agencia Estatal de Investigación
OpenAlex ID: W4415304952